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teacher candidates from a university in the Black Sea Region as the data collecting tool. The data was analyzed with statistical software (SPSS 13.0) in terms of average mean, standard deviation and t-tests. Current findings show that .... The articl
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THE THAI JOURNAL OF VETERINARY MEDICINE Office: Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330 Thailand Tel. 66(2)- 218 9676 Fax. 66(2)- 218 9677 Advisory Committee: Prof. Dr. Mongkol Techakumphu Dean
Dr. Yukol Limleamthong President of the Veterinary Council of Thailand
Assoc. Prof. Dr. Benjamas Patamalai Associate Dean (Research Affairs)
Assoc. Prof. Dr. Achariya Sailasuta President of the Thai Veterinary Medical Association under the Royal Patronage Assoc Prof. Dr. Wimon Pothiwong President of Chulalongkorn University Veterinary Alumni Association
Miss Pringsri Ingkaninun Assistant Dean (Physical Resource Management Affairs)
Andrew Ponter (France) Andrzej Madej (Sweden) Eileen L. Thacker (USA) Elisabeth Persson (Sweden) Han-Soo Joo (USA) Karen L. Keller (USA) Oliver Sparagano (UK) Stanley H. Done (UK) Stig Einarsson (Sweden) Takashi Aoki (Japan) Teresa Y. Morishita (USA)
The Thai Journal of Veterinary Medicine Vol. 41 No. 1 March 2011 Editor's Note
Emerging Drug-resistant Superbugs
Rungtip Chuanchuen Reveiw Article From Microsurgery to Single Blastomere Biopsy for ES cell Establishment Chanchao Lorthongpanich, Chuti Laowtammathron, Rangsun Parnpai Original Article Fas Ligand in Swamp Buffalo Oviduct during Follicular and Luteal Phases Paisan Tienthai, Pongpisut Chivacharern, Kriengyot Sajjarengpong, Pornchalit Assavacheep The Virulence of Thai Isolated Mycoplasma gallisepticum in Challenged Embryonated Eggs Somsak Pakpinyo, Suwarak Wanaratana, Sarawoot Mooljuntee Clinical Study on the Treatment of Piroline against Bovine Mastitis Jian-Ping Liang, Bao-Cheng Hao, Xue-Hong Wang, Zhi-Ting Guo, Wen-Zhu Guo, Ruo-Fong Shang, Lei Tao, Yu Liu, Zhao-Zhau Li, Lan-Ying Hua, Shu-Yang Wang Chronic Toxicity Study of Garcinia mangostana Linn. pericarp Extract Songpol Chivapat, Pranee Chavalittumrong, Prapai Wongsinkongman, Chada Phisalpong, Anudep Rungsipipat Genetic Characterization of Porcine Epidemic Diarrhea Virus (PEDV) Isolates from Southern Vietnam during 2009-2010 Outbreak Do Tien Duy, Nguyen Tat Toan, Suphasawatt Puranaveja, Roongroje Thanawongnuwech Immunolocalization of Estrogen Receptor beta, Androgen Receptor and Ki-67 Protein in Testicular Tissues of Unilateral Cryptorchidism Boar Sukanya Manee-in, Pinthira Thiengthiantham, Chanlika Prommapan, Kampon Kaeoket Improvement of Normal Fertilization Rate and Embryo Development by Reduction of Sperm: Oocyte ratio during in vitro Fertilization in Pig Sasithorn Panasophonkul, Theerawat Tharasanit, Panida Chanapiwat, Mongkol Techakumphu Differentiation Potentials of Canine Bone Marrow Mesenchymal Stem Cells Theerawat Tharasanit, Nawapen Phutikanit, Chalika Wangdee, Kumpanart Soontornvipart, Sasijaras Tantrajak, Theerayuth Kaewamatawong, Junpen Suwimonteerabutr, Pitt Supaphol, Mongkol Techakumphu Anticlastogenic Effect of Asiatic Pennywort and Indian Mulberry using Rodent Erythrocyte Micronucleus Assay Piengchai Kupradinun, Anong Tepsuwan, Wannee R. Kusamran Short Communication Seroprevalence of Antibodies against Bartonella hensalae Infection in Cats and Dogs along the Northern Borders of Thailand Decha Pangjai, Soichi Maruyama, Sumalee Boonma, Wimol Petkanchanapong, Wattanapong Wootta, Pathom Sawanpanyalert Presence of Infectious Pancreatic Necrosis Virus on Rainbow Trout (Oncorhynchus mykiss) by Histopathology, ELISA and RT-PCR Gabriel Aguirre-Guzmán, Ned Iván de la Cruz-Hernández, Jesús Genaro Sánchez-Martínez The Investigation of the Relations between Insulin-liked Growth Factor-I and Body Weight and between Insulin-liked Growth Factor-I and Sex in Young Cats Wei-Yau Shia, Anchana Songkaew, Sasisopa Singhanetr, Chih-Chung Chou, Wei-Ming Lee The Activity of Plasma Matrix Metalloproteinase-9 as a Marker to Reflect the Tissue Level of Mammary Glands Tumor in Dogs Wei-Yau Shia, Shih-Ming Liu, Chun-Sheng Lee, Jian-Liang Lin, Chih-Chung Chou, Yumi Yuasa, Shih-Chieh Chang, Wei-Ming Lee Diagnostic Forum ECG Quiz Chollada Buranakarl, Winai Chansaisakorn Ophthalmology Snapshot Nalinee Tuntivanich Ultrasound Diagnosis Phiwipha Kamonrat What is your Diagnosis Pranee Tuntivanich, Suwicha Chuthatep
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4 INSTRUCTIONS TO AUTHORS The Thai Journal of Veterinary Medicine publishes articles reporting interdisciplinary investigations concerning veterinary and animal sciences, at all levels of resolution, from basic to clinical, molecular to behavioral, and opinions that are of general interest to the broad community of veterinarians and biological scientists. Clinical or pathological investigations, protocols and reviews will also be considered for publication if they provide significant insight into the structure or function, the pathophysiology of a disease, or its treatment. In the Journal’s Table of Contents, published articles will be shown under one of the appropriate Section titles listed below SECTIONS Editorials A limited amount of space will be available for comments about important scientific points or subjects of topical interest, and will be by invitation only. Reviews will be either by invitation, or submission. The latter will be reviewed by experts in the same manner as other submitted manuscripts. Original Articles should be novel research findings and provide strong evidence for the conclusions. The manuscripts suitable for publication in TJVM should be of extreme importance to scientists in the field as well as interesting to researchers in other disciplines. Short Communications These are short communications that describe outstanding new discoveries. This decision will be based on whether the paper reports particularly important findings that are likely to have a high impact in the field of work. Clinical or Pathological Reports These are short reports of original clinical or pathological findings whose importance mean that they will be of interest to veterinarians. Diagnostic Forum is a regular feature of TJVM. This includes Ultrasound Diagnosis, ECG Quiz, Ophthalmology Snapshot and What Is Your Diagnosis, all of which will be by invitation only. SUBMISSION POLICY Submission of a paper to TJVM is understood to imply that it deals with original material not previously published, and that it is not being considered for publication elsewhere. Please write your text in good English (American or British usage is accepted, but not a mixture of these). The layout and style should adhere strictly to the instructions given under “Organisation of the Article”. Two copies of the manuscripts should be submitted to the Editorial Board, The Thai Journal of Veterinary Medicine, Chulalongkorn University, Bangkok 10330, Thailand. Fax: 02 218 9677. The final version of the manuscript including all figures and tables should be submitted in digital form. No revisions or updates will be incorporated after the article has been accepted and sent to the Publisher (unless approved by the Editorial Board). The Editorial Board reserves the right to reject any manuscript deemed unsuitable for publication in TJVM. Upon acceptance, a letter will be sent to the corresponding Author confirming receipt of the manuscript. Accepted manuscripts become the property of TJVM. The Journal reserves the copyright, and no published material may be reproduced or published elsewhere without written consent from the Journal. If excerpts from other copyrighted works are included, the
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Emerging Drug-resistant Superbugs Rungtip Chuanchuen Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok, 10330 Thailand Antimicrobial agents are drugs, chemicals and substances that either kill or inhibit the growth of microorganisms including susceptible bacteria (Gilbert and McBain, 2003). Unfortunately, bacteria could change in a way that they can survive in the presence of the antimicrobials. Susceptible bacteria get eliminated while resistant clones stay alive, continue to multiply and cause more damages. When bacteria resist the effects of an antimicrobial agent, it is referred to as resistant. If a bacterial strain is exceedingly resistant to almost every known and antibiotics available, it is informally called “a superbug” or “a super bacterium” implying a super fighter against antimicrobial agents (Reinhardt, 2010). While bacteria could be harmless and sometimes helpful, superbugs are mostly pathogenic bacterial strains and infections with superbugs usually result in increased severity of diseases, prolonged hospitalized stay, higher cost of treatment and increased mortality rate. Existing-clinical outcomes demand the new alternative treatments, particularly new medications that could overcome the development of multiresistance in superbugs. Fearfully, superbugs are so talent that they can build up or acquire a resistance to most antimicrobials available in the markets including novel drugs designed to treat bacteria that have already become resistant to former-generation drugs and then, become stronger. There are a variety of reasons for the increasing emergence of antimicrobial resistance among bacteria and the main reason is antimicrobial usage in both humans and animals. In humans, antimicrobial agents are often inappropriately taken due to the expectation of rapid infection cure. In many countries around the world, several antibiotics are sold over the counter without a prescription and inappropriate prescribing of antibiotics has been simply issued by physicians. All these misbehaviors enhance the growing of resistance among bacteria leading to ineffective treatment. Similarly, the antimicrobial substances have been widely used in food-animal production for three main purposes including treatment of diseases, prevention of infection and promoting of growth. The latter has appeared to make up the largest portion of total veterinary antimicrobial uses and mainly contribute to the widespread dissemination of antimicrobial resistance among bacteria. Antimicrobial use in farm animals evidently poses superbug risk to humans. This is based on the fact that the greater the length of antimicrobial exposure time, the greater the chance of development of resistance. When the board spectrum
antimicrobial agents are used and other bacteria are killed, superbugs can grow better and their infection rate will be promoted. As a rule of thumb, whenever an antimicrobial agent is used, it drives forward the development of resistant bacteria and of course, superbugs. Up to date, superbugs have emerged around the world and most of them appear as the life threatening pathogens. These emerging-deadly superbugs include: 1. Escherichia coli and Klebsiella pneumoniae harboring New Delhi metallo β-lactamase (NDM-1) The NDM-1 gene encodes β-lactamase enzymes carbapenemases conferring resistance to a board range of β-lactams. The enzymes are commonly produced by Gram-negative bacteria, in particular E. coli and K. pneumoniae (Paterson, 2006). The NDM-1 gene was first detected in a K. peumoniae isolate from a Swedish patient who travelled to New Delhi in 2008 (Yong et al., 2009) and has emerged as a newest superbug quickly arisen as one of the most feared pathogens. The NDM-1 gene is horizontally transferred and has spread to many countries worldwide in a short time. Special awareness has been raised against the NDM-1 carrying E. coli strains that could resist to cabapenems the most powerfulantibiotic group currently available (Queenan and Bush, 2007). 2. Methicillin-resistant Staphylococcus aureus (MRSA) MRSA is the most recognized superbugs that have become very common in many hospitals throughout the world. Methicillin was introduced in 1959 for treatment of penicillin-resistant S. aureus and soon later, MRSA was first detected in 1961 (Ippolito et al., 2010). At first, MRSA was limited to hospitals and healthcare settings and so known as hospitalacquired MRSA infection. Since the late 1990s, the MRSA epidemiology encounter a major change that is the emergence of community-acquired MRSA strains with rapid spread and causing fatal diseases (Ippolito et al., 2010). Most MRSA strains exhibit resistance to multiple drugs including penicillin, methicillin, tetracycline, erythromycin and vancomycin (de Lencastre et al., 1996; Smith et al., 1999). 3. Multidrug resistant Strptococcus and Enterococcus Two major-streptococcal superbugs posing lethal risk include S. pyrogenes Group A and S. pneumoniae. S. pyrogenes Group A or Strep A is notoriously known as a flesh-eating bacterium due to its ability to produce toxins that cause necrotizing fasciitis and the most rapid treatment is removing the damage tissues. S. pneumonia resistant to penicillin
Thai J Vet Med. 2011. 41(1): 7-9
8 and other β-lactams have increasingly caused sickness and death worldwide (Schrag et al., 2001). This superbug has additionally developed resistance to trimethoprim, sulfmethoxazole, azithromycin, tetracyclines, minocycline and fluoroquinolones (Hoenigl et al., 2010). Multi-resistant E. faecium is another superbug that is frequently found in hospitals. This superbug is normally not harmful. It will become very dangerous and deadly when enters urinary tract and open wound. Vancomycin-resistant Enterococcus (VRE) is prominently known as the leading cause of urinary tract infection and meningitis (Sood et al., 2008). 4. Multidrug-resistant P. aeruginosa and Acinetobactor baumannii (MRAB) Both P. aeruginosa and A. baumannii are opportunistic pathogens that are the important causes of hospital acquired infection. These superbugs have become predominant in most intensive care settings and some strains cannot be treated with any systemic antibiotics available. P. aeruginosa has been disreputably known for its highly intrinsic resistance to multiple drugs due to the synergy of low permeability cell and the expression of multidrug efflux systems envelope. This superbug has a great ability to form biofilms that slow down the intracellular penetration of antimicrobial molecules once again leading to resistance to various antimicrobials (Costerton et al., 1999). Infection with A. baumannii is commonly associated with patients with immunosuppression and having invasive, of which death rate is high up to 80% (Turton et al., 2006). The apparent resistance in the MRAB strain has limited drug choice for treatments especially carbapenems. 5. Multidrug-resistant and Extensively drug-resistant tuberculosis (MDR- and XDR-TB) Tuberculosis is a dreadful disease that is acquired by inhalation. MDRTB is caused by strains of Mycobacterium tuberculosis and has become a leading cause of death among HIVinfected individuals. This superbug is resistant to one of two first-line antibiotics isoniazid and rifampicin and can be treated with second-line drugs (i.e. kanamycin, amikacin and capreomycin) that are more expensive and have more side-effects (Jugheli et al., 2009). XDR-TB is a subset of MDR-TB that is additionally resistant to fluoroquinolones and one of the powerful second-line drugs (Jain and Dixit, 2008). Emergence of XDR-TB has raised a serious concern of restricted antitubercular drugs for a future TB treatment. 6. Multidrug-resistant Clostridium difficile C. difficile is a commensal bacterium in human intestine that does not cause any significant disease when it is present in small number. Once the normal intestinal flora is disrupted in particular by a board spectrum antibiotic, C. difficile will overgrow and produce several toxins leading to severe illness i.e. pseudomembranous colitis (PMC). This phenomenon usually takes place after long-term hospitalization with use of clindamycin, ampicillin, amoxicillin, and cephalosporins (Mylonakis et al., 2001). This superbug is considered the most serious cause of antibiotic-associated diarrhea and informally called “a spore spread superbug” (Lawley et al., 2009). Its spores are heat-tolerant and resistant to alcohol and several routine disinfectants (except bleach). To date, C. diffile strains resistant to many front line
Chuanchuen R. / Thai J Vet Med. 2011. 41(1): 7-9 antimicrobial agents i.e. clindamycin, ciprofloxacin, levofloxacin, metronidazole, linezolid have been isolated (Loo et al., 2005; Rupnik et al., 2009). 7. Multidrug-resistant E. coli 025b-ST131 Multiresistant E. coli strains are frequently found and do not seem to provoke much excitement until an outbreak occurs. Whenever this superbug is spread, serious public health problem will arise and some death will be always reported. The well-known E. coli superbug produces Extended-Spectrum β-Lactamase (ESBL) and is almost untreatable (Hsieh et al., 2010). A new-virulent drug resistant strain of E. coli serogroup 025b sequence type 131 (ST131) has emerged as an important extraintestinal pathogen that is usually contaminated with animal feces, transferred to food of animal origin and also associated with infection of gastrointestinal tract. ST131 exhibits resistance to many β-lactams, which is mostly related to CTX-M-15 the most commonlyidentified CTX-M mediating resistance to penicillin, cephalosporins and monobactams (Johnson et al., 2010). This superbug also commonly exhibits resistance to antibiotics in groups of fluoroquinolones, sulphamethoxazole and trimethoprim (Johnson et al., 2009; Johnson et al., 2010). 8. Multidrug-resistant Salmonella Highly drug resistant-Salmonella has increased and is now recognized as a lethal-food poisoning superbug commonly associated with meat and meat products. In general, transfer of resistance trait in this superbug is very efficient through a mobile genetic elements class 1 integrons and several conjugative R-plasmids (Khemtong and Chuanchuen, 2008; Wannaprasat et al., 2011). Concerns are now growing on a Salmonella superbug, S. Typhimurium DT104, which exhibits resistance to at least four antibiotics and is now becoming more resistant. Arising of resistance among bacteria is considered a dynamic process. The constantly-evolving resistance mechanisms enforce scientists and experts to continually develop new antimicrobial agents. However, it does not seem to be enough to keep up with the rapid development of bacterial resistance. Emergence of deadly superbugs raises a great concern that there will be eventually no antimicrobials effective for infection treatment and calls for the attention that it is now a crucial time to develop strategies to preserve the efficacy of antimicrobial agents for future generations. In fact, the rigorous hygiene may be the best way to stop superbugs from spreading and producing damages since multidrug-resistant bacteria could be simply controlled by the comprehensive sanitation e.g. strictly cleaning and disinfecting of all medical equipment and rooms, thoroughly hand washing, early checking the patient for superbugs. It comes to the fact that the patients that use the most antimicrobials are livestock. Even though antimicrobial agents clinically important to human medicine are suggested to be used in farm animals only for medical purposes, this is still far away from what actually happens in today-veterinary practices. As it is necessary to use antimicrobial agents to protect both animal and human health, awareness of using these substances should be priorities in both human and veterinary medicine.
Chuanchuen R. / Thai J Vet Med. 2011. 41(1): 7-9. References Costerton, J.W., Stewart, P.S. and Greenberg, E.P. 1999. Bacterial biofilms: a common cause of persistent infections. Science. 284: 1318-1322. de Lencastre, H., de Lencastre, A. and Tomasz, A. 1996. Methicillin-resistant Staphylococcus aureus isolates recovered from a New York City hospital: analysis by molecular fingerprinting techniques. J Clin Microbiol. 34(9): 2121-2124. Gilbert, P. and McBain, A.J. 2003. Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance. Clin Microbiol Rev. 16(2): 189-208. Hoenigl, M., Fussi, P., Feierl, G., Wagner-Eibel, U., Leitner, E., Masoud, L., Zarfel, G., Marth, E. and Grisold, A.J. 2010. Antimicrobial resistance of Streptococcus pneumoniae in southeast Austria, 1997-2008. Int J Antimicrob Agents. 36(1): 24-27. Hsieh, C.J., Shen, Y.H. and Hwang, K.P. 2010. Clinical implications, risk factors and mortality following community-onset bacteremia caused by extendedspectrum beta-lactamase (ESBL) and non-ESBL producing Escherichia coli. J Microbiol Immunol Infect. 43(3): 240-248. Ippolito, G., Leone, S., Lauria, F.N., Nicastri, E. and Wenzel, R.P. 2010. Methicillin-resistant Staphylococcus aureus: the superbug. Int J Infect Dis 14(Suppl 4): S7-S11. Jain, A. and Dixit, P. 2008. Multidrug-resistant to extensively drug resistant tuberculosis: What is next? J Biosci. 33(4): 605-616. Johnson, J.R., Johnston, B., Clabots, C., Kuskowski, M.A. and Castanheira, M. 2010. Escherichia coli sequence type ST131 as the major cause of serious multidrugresistant E. coli infections in the United States. Clin Infect Dis. 51(3): 286-294. Johnson, J.R., Menard, M., Johnston, B., Kuskowski, M.A., Nichol, K. and Zhanel, G.G. 2009. Epidemic clonal groups of Escherichia coli as a cause of antimicrobial-resistant urinary tract infections in Canada, 2002 to 2004. Antimicrob Agents Chemother. 53(7): 2733-2739. Jugheli, L., Bzekalava, N., de Rijk, P., Fissette, K., Portaels, F. and Rigouts, L. 2009. High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene. Antimicrob Agents Chemother. 53(12): 5064-5068. Khemtong, S. and Chuanchuen, R. 2008. Class 1 Integrons and Salmonella genomic island 1 among Salmonella enterica isolated from poultry and swine. Microb Drug Resist. 14(1): 65-70. Lawley, T.D., Clare, S., Walker, A.W., Goulding, D., Stabler, R.A., Croucher, N., Mastroeni, P., Scott, P., Raisen, C., Mottram, L., Fairweather, N.F., Wren, B.W., Parkhill, J. and Dougan, G. 2009. Antibiotic treatment of Clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts. Infect Immun. 77(9): 3661-3669. Loo, V.G., Poirier, L., Miller, M.A., Oughton, M., Libman, M.D., Michaud, S., Bourgault, A.M., Nguyen, T., Frenette, C., Kelly, M., Vibien, A., Brassard, P., Fenn, S., Dewar, K., Hudson, T.J., Horn, R., Rene, P., Monczak, Y. and Dascal, A. 2005. A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med. 353(23): 2442-2449.
9 Mylonakis, E., Ryan, E.T. and Calderwood, S.B. 2001. Clostridium difficile--Associated diarrhea: A review. Arch Intern Med. 161(4): 525-533. Paterson, D.L. 2006. Resistance in gram-negative bacteria: Enterobacteriaceae. Am J Infect Control. 34(5 Suppl 1): S20-S28; discussion S64-S73. Queenan, A.M. and Bush, K. 2007. Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev. 20(3): 440-458. Reinhardt, D. 2010. Antibiotic resostance superbugs-bacteria as a superfighter. http://www.suite101.com/content/superbugs-they-infect-and-can-kill--superbug-facts-to-knowa272844. Rupnik, M., Wilcox, M.H. and Gerding, D.N. 2009. Clostridium difficile infection: New developments in epidemiology and pathogenesis. Nat Rev Microbiol. 7(7): 526-536. Schrag, S., Beall, B. and Dowell, S. 2001. Resistant pneumococcal infections: the burden of disease and challenges in monitoring and controlling antimicrobial resistance (WHO/CDS/CSR/DRS/2001.6): 35 pp. Smith, T.L., Pearson, M.L., Wilcox, K.R., Cruz, C., Lancaster, M.V., Robinson-Dunn, B., Tenover, F.C., Zervos, M.J., Band, J.D., White, E. and Jarvis, W.R. 1999. Emergence of vancomycin resistance in Staphylococcus aureus. N Engl J Med. 340(7): 493501. Sood, S., Malhotra, M., Das, B.K. and Kapil, A. 2008. Enterococcal infections & antimicrobial resistance. Indian J Med Res. 128(2): 111-121. Turton, J.F., Kaufmann, M.E., Gill, M.J., Pike, R., Scott, P.T., Fishbain, J., Craft, D., Deye, G., Riddell, S., Lindler, L.E. and Pitt, T.L. 2006. Comparison of Acinetobacter baumannii isolates from the United Kingdom and the United States that were associated with repatriated casualties of the Iraq conflict. J Clin Microbiol. 44(7): 2630-2634. Wannaprasat, W., Padyngtod, P. and Chuanchuen, R. 2011. Class 1 integrons and virulence genes in Salmonella enterica isolates from pork and humans. Int. J Antimicrob Agents. In press. Yong, D., Toleman, M.A., Giske, C.G., Cho, H.S., Sundman, K., Lee, K. and Walsh, T.R. 2009. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother. 53(12): 5046-5054.
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From Microsurgery to Single Blastomere Biopsy for ES cell Establishment Chanchao Lorthongpanich1* Chuti Laowtammathron2 Rangsun Parnpai3*
Abstract Inner cell mass (ICM) is an important source of embryonic stem (ES) cells. There are several methods that have been developed to increase the efficiency of ICM cell isolation as well as improve the ES cells derivation rate. In conventional ICM isolation methods, the methods currently in use destroy trophectoderm cells of blastocyst stage embryos in order to release the ICM cell clumps. These conventional methods are considered embryo destruction methods as the embryos will be unable to survive after ICM is removed. Recently, an alternative method was reported using single biopsied blastomeres of earlier stage embryos as a source of ES cells. This novel method provides a new, ethically positive option that avoids destroying the embryos. This brief review of the techniques involved in ICM isolation and ES cells establishment, along with methodological comparisons, outlines the development of each technique, which may be used as a resource for choosing a suitable procedure for future experiments. Keywords: blastocyst, blastomere, embryonic stem cell, immunosurgery, inner cell mass, microsurgery 1Mammalian
Development Laboratory, Institute of Medical Biology, Biopolis, Singapore. Cell and Developmental Biology Laboratory, Genome Institute of Singapore, Biopolis, Singapore. 3Embryo Technology and Stem Cell Research Center, School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima, Thailand. Corresponding author E-mail: [email protected], [email protected] 2Stem
Thai J Vet Med. 2011. 41(1): 11-22.
Lorthongpanich C. et al. / Thai J Vet Med. 2011. 41(1): 11-22.
บทคัดย่อ จากเทคนิคจุลศัลยกรรมสู่เทคนิคการใช้เซลล์ตวั อ่อนเพียงหนึ่งเซลล์สําหรับการผลิตเซลล์ต้น กําเนิดตัวอ่อน จันทร์เจ้า ล้อทองพานิชย์ 1* ชุติ เหล่าธรรมธร 2 รังสรรค์ พาลพ่าย 3* อินเนอร์เซลล์แมส (ICM) เป็นแหล่งของเซลล์ที่สําคัญที่สุดในการผลิตเซลล์ต้นกําเนิดตัวอ่อน การสกัดเซลล์ ICM ออกจากตัวอ่อน สามารถทําได้หลายวิธี จวบจนปัจจุบันหลายวิธีได้ถูกพัฒนาเพื่อให้ได้เซลล์ ICM ที่มีประสิทธิภาพดีและสามารถเพิ่มอัตราความสําเร็จในการ ชักนําให้เป็นเซลล์ต้นกําเนิดตัวอ่อนได้ ทั้งนี้วิธีสกัดเซลล์ ICM แบบดั้งเดิมเป็นเทคนิคที่ต้องทําลายตัวอ่อน เนื่องจากตัวอ่อนจะไม่สามารถ พัฒนาต่อไปได้เมื่อ ICM ถูกสกัดออกไป แต่ล่าสุดมีการพัฒนาเทคนิคใหม่ขึ้นมาเป็นอีกหนึ่งทางเลือกที่จะทําให้ผลิตเซลล์ต้นกําเนิดตัวอ่อนได้ โดยไม่ต้องทําลายตัวอ่อน เนื่องจากจะใช้เซลล์ตัวอ่อนที่ระยะต้นๆ ของการพัฒนาเพียงหนี่งเซลล์มาใช้เป็นแหล่งของเซลล์ต้นกําเนิดตัวอ่อน แทนการใช้ ICM จากตัวอ่อน ระยะบลาสโตซีส เทคนิคการผลิตเซลล์ต้นกําเนิดตัวอ่อนจากเซลล์ตัวอ่อนระยะต้นเพียงหนึ่งเซลล์นี้สามารถลด ข้อจํากัดทางจริยธรรมลงได้ เนื่องจากตัวอ่อนที่ถูกสกัดเซลล์ออกมาเพียงหนึ่งเซลล์จะยังสามารถพัฒนาต่อไปได้เช่นเดียวกับตัวอ่อนปกติ ใน เอกสารฉบับนี้ได้รวบรวมเทคนิคต่างๆ ที่เกี่ยวข้องกับการสกัดเซลล์ ICM และผลิตเซลล์ต้นกําเนิดตัวอ่อน นอกจากนี้ยังมีข้อมูลสําหรับ เปรียบเทียบแต่ละเทคนิค รวมถึงการพัฒนาเทคนิคแต่ละวิธีจากอดีตจนถึงปัจจุบันเพื่อจะได้เป็นประโยชน์ ในการวางแผนการทดลองของผู้ที่ สนใจต่อไปในอนาคต คําสําคัญ: บลาสโตซีส บลาสโตเมียร์ เซลล์ต้นกําเนิดตัวอ่อน immunosurgery อินเนอร์เซลล์แมส microsurgery 1 Mammalian Development Laboratory, Institute of Medical Biology, Biopolis, Singapore. 2 Stem Cell and Developmental Biology Laboratory, Genome Institute of Singapore, Biopolis, Singapore. 3 Embryo Technology and Stem Cell Research Center, School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima, Thailand. *ผู้รับผิดชอบบทความ E-mail: [email protected], [email protected] Introduction The blastocyst contains trophectodermal (TE) and inner cell mass (ICM) cells. TE cells develop into the placenta after the blastocyst implants to the endometrium layer of a mother’s uterus. ICM cells develop into three embryonic germ layers and a yolk sac. The three embryonic germ layers eventually incorporate into a fetus. In other words, the ICM is differentiated into the three embryonic germ layers of the fetus. Therefore, the ICM, which can only be observed when the embryo develops to the blastocyst stage, is the only source for establishing an ES cell. High quality blastocysts with distinct ICM cells are the most appropriate for ES cell establishment. In general, one blastocyst contains only one ICM. If the ICM is removed to establish an ES cell line then the blastocyst has no further chance to implant and develop into a fetus. Since 1981, there have been several methods of ICM cells isolation that have been developed, but among those techniques immunosurgery seem to be the most favored technique among researchers because it is the most
effective and the simplest method to isolate the clear ICM cells (Solter and Knowles, 1975; Martin,1981; Piedrahita et al., 1990; Chen et al., 1999; Anderson et al., 1994; Thomson et al., 1995; Moore and Piedrahita, 1997; Thomson et al., 1998; Reubinoff et al., 2000; Suemori et al., 2001; Vrana et al., 2003; Cowan et al., 2004; Heins et al., 2004; Brevini et al., 2005; Ock et al., 2005; Mateizel et al., 2006; Mitalipov et al., 2006; Shiue et al., 2006). This could explain why almost all ES cell lines have been established from the ICM cells of blastocyst embryos since then. Unfortunately, immunosurgery required animal products in the process, such as anti-sera and guinea pig complement, which adds a risk of pathogen contamination, especially when using this technique to establish human ES cell lines. Recently, a novel method for ES cell establishment has been developed by using single blastomeres of early stage embryos as a source of ES cells (Chung et al., 2006). It has been introduced as another method of choice to address ethical issues, especially those of embryo destruction. Additionally, it could be used as a method for establishing the patient specific ES cells. This review will summarize the methods involved in ICM cells isolation including
Lorthongpanich C. et al./ Thai J Vet Med. 2011. 41(1): 11-22. the techniques that destroy embryos (conventional techniques) and those that do not (novel methods). However, each procedure has specific characteristics that need to be considered before their use with your embryos.
Since 1972, ICMs have been isolated by mechanical techniques (Gardner, 1972). Several methods have since been established, such as immunosurgery, calcium ionophore (A23187), whole or partial blastocyst culture, laser dissection and digestion methods. Each method has been used in ICM isolation for ES cell establishment. However, these methods are considered embryo destruction techniques, which clearly raise ethical issues. Yet some of these techniques such as immunosurgery and whole or partial embryo culture have widely still been in use. 1.1. Microsurgery This technique was first reported by Gardner (1972) for TE and ICM cells separation in his investigation of the function of TE and ICM cells of mouse blastocysts (Gardner, 1972). The separation was performed by micromanipulator. The blastocysts were placed in a drop of medium hanging from the coverslip of a chamber filled with heavy liquid paraffin. Then, the blastocysts were held by suction pipette against the underside of the coverslip of the hanging drop. A piece of micro-blade was attached and arranged vertically on one manipulator unit, so that the micro-blade surface was parallel with the coverslip of the chamber. Then the micro-blade was slowly moved parallel to the ICM cells surface to cut the TE cells (Gardner, 1972; Rossant, 1975). This method was later modified for isolating ICM cells from blastocysts and used as a source of ES cells. The manipulation technique was modified by using only two fine needles for ICM dissection under stereomicroscope which was much more practical and easier to perform. It worked very well in several in vivo produced embryos such as cat (Yu et al., 2008) and a human embryo (Kim et al., 2005) because the in vivo produced embryos had more distinct ICM cells than the embryos produced in vitro. Later in 2007, Strom and colleagues reported a new mechanical technique for ICM isolation by using a specially made flexible tungsten needle, with a diameter of 0.125 mm. The tip was thin and sharpened using electrolysis. Another blunter needle was used to hold the blastocyst during the cutting out of the ICM. Both needles were fixed to hand-pieces of pencil-thickness for manual operation under stereomicroscope. The blastocyst was moved to the operation drop then drawn out with the needle so that the blastocyst became attached to the surface of the well. With the blastocyst attached to the plastic, it was possible to make a hole in the zone pellucida with the needle to open up the blastocyst and by making two to three cuts, to remove the ICM from the trophectoderm. The procedure took about two to three minutes per embryo (Strom et al., 2007).
13 1.2. Immunosurgery Immunosurgery can be used to obtained large quantities of pure ICM masses in relatively short periods of time (Solter and Knowles, 1975). It relies on the compatibility of antibody to the cell surface antigen of trophoblastic cells of the blastocyst stage embryo. This compatibility causes a selective killing of trophoblastic cells after the complement is added into the system. Briefly, the blastocysts are cultured with a proper concentration of antigen for about 30 minutes (Solter and Knowles, 1975). The antigen is usually derived from serum of rabbit anti spleenocytes of the target embryo’s species. Then the blastocysts are subsequently washed several times to discard the unbound antibodies before incubation with a suitable concentration of complement for 15-30 minutes. During the incubation with complement, trophoblastic cells swell, and finally loose their semipermeability. Pilz and colleagues (1970) suggested that the mouse blastocyst was impermeable for molecules larger than 40A°. This makes the passage of an immunoglobulin molecule (antibody) with a larger diameter impossible. Therefore, to intensively wash out the unbound antibodies will secure ICM cells, because there will be no antibodies left over to bind with the antigen on the ICM cells surface after the TE cells are destroyed in the complement. To do so, only TE cells will be destroyed, thus leaving the ICM intact. One of the advantages of this method is that it allows the recovery of many ICMs without the risk of mechanical damage that might occur in a microsurgical method (Solter and Knowles, 1975). To assess the purity of ICM cells after immunosurgery, Handyside and Barton (2007) studied the failure to detect fluorescent-conjugate antibodies in IgGs or trophoblastic-type outgrowths in vitro and found that the dissected ICMs showed a negative result. Moreover, they found that the protein synthetic profile of these ICMs was similar to microsurgically dissected ICMs, and in particular, trophoblast specific spots were absent. Additionally, when the ICM cells were transferred to the uterus of the pseudopregnant mice for evaluation of the implantation capacity, they only found the ICM derived tissues. This finding demonstrates the lack of TE cell contamination and functional viability of these ICMs. In 1981, Martin, G.R. established the first ES cell lines by using immunosurgery as a method for ICM cells dissection before culturing ICM cells in teratocarcinomas conditioned medium. Several days after culture, the ICMs showed remarkable resemblance to pluripotent morphologies as embryonal carcinoma (EC) stem cell lines. Martin named the pluripotent cell lines derived from ICM cells as embryonic stem (ES) cells to denote their origin directly from embryos and to separate them from EC cells which were derived from teratocarcinomas (Martin, 1981). After the success of the first mouse ES cell lines established by immunosurgery, several attempts in other animal species were reported such as pig (Piedrahita et al., 1990; Anderson et al., 1994; Moore and Piedrahita, 1997; Chen et al., 1999; Brevini et al., 2005; Ock et al.,
14 2005; Shiue et al., 2006), monkey (Thomson et al., 1995; Suemori et al., 2001; Vrana et al., 2003; Mitalipov et al., 2006), and human (Thomson et al., 1998; Reubinoff et al., 2000; Cowan et al., 2004; Heins et al., 2004; Mateizel et al., 2006). Currently, immunosurgery is a commonly used method for establishing ES cells from ICM of blastocyst stage embryo. 1.3. Calcium Ionophore A23187 Calcium ionophore A23187 is a monocarboxylic acid antibiotic, specifically a divalent cation. It increases intracellular ionized calcium (Reed and Lardy, 1972) and also includes mitogenic effects on lymphocytes (Freedman, Raff & Gomperts, 1975; Hesketh et al. 1977), inhibited morphological changes in cells induced by dibutyryl cyclic AMP (cAMP; Henneberry et al., 1975), releases histamine from most cells (Foreman et al., 1973), causes prevention of retinal orientation in developing eyes of Xenopus laevis (Rose and Loewenstein, 1975; Jacobson, 1976), and chemically activated unfertilized eggs such as mouse ( Hagemann et al., 1994; Uranga et al., 1996), cat (Grabiec et al., 2007), pig (Wang et al., 1998) and bovine (Liu et al., 1998; Chung et al., 2001; Xu and Yang, 2001; Sedmikova et al., 2003). It also can be used as one of the activation reagents in cloned embryo development in several species such as bovine (Milazzotto et al., 2008), buffalo (Saikhun et al. 2003) and human (Heindryckx et al., 2007). For ICM isolation, calcium ionophore A23187 was accidentally found during the course of experiments to detect the mitogenic action of A23187 on blastocysts. Surani and colleagues (1978) found that 2x10-5 M calcium ionophore (A23187) caused selective lysis of trophectoderm cells and occurred after approximately 30 min following their swelling and vesiculation, but the ICM apparently remained intact (Surani et al., 1978). Using calcium ionophore A23187 to destroy TE cells is morphologically identical to the lysis of the cell observed after treatment of blastocysts with antibodies and complement similar to that used in the immunosurgery method. The ICM recovery rate after the late expanded blastocysts were treated with ionophore was 100%, but the recovery rate was lower when early stage blastocysts were used (75-82%; Surani et al., 1978). Calcium ionophore A23187 was used in some later reports for ICM isolation of rodent (Harlow and Quinn, 1979; 1980; Piedrahita et al., 1990) and ovine embryos (Piedrahita et al., 1990). The mechanism of TE cells lysis after being treated with calcium ionophore A23187 is still not clear. It can be due to an osmotic phenomenon which can be explained by either the suggestion that sodium (Na+) flows into the cells more quickly than the outward flow of potassium (K+), that the influx of water then causes swelling and lysis of cells (Green et al., 1959) or the ionophore induces the uncontrolled flux of carbonic anhydrase II (Reed and Larde, 1972) followed by swelling and vesiculation after the entry of water into the cells. This method, however, is not generally used for ICM cell isolation today. This might be because of the uncontrollable osmotic action and mechanism of destroying the TE cells are not clear and, as mentioned above, the calcium ionophore A23187 can be used in several ways which might
Lorthongpanich C. et al. / Thai J Vet Med. 2011. 41(1): 11-22. generate side effects or affect the success rate of ES cells establishment. 1.4. Whole Blatocyst or Partial Blastocyst Culture The whole blastocyst and partial blastocyst culture methods seem to be the most advantageous to produce pathogen-free ES cell lines especially for human ES cells, because of the absence of animal products such as antibody and complement, which are used in immunosurgery methods. Whole blastocyst culture has been used for ES cell establishment since 1981 when Evan and Kaufman (1981) cultured the delayed blastocysts in culture drops to let the embryos attach to the feeder cells. After attachment, TE cells grew out and differentiated to giant cells, where as ICM cells formed into egg cylinder shapes. The ICM cells were picked off and dispersed by trypsin treatment and reseeded onto feeder cells. By using this technique, they established 15 pluripotent cell lines from an independent embryo (Evans and Kaufman, 1981). This method has been used in particularly with animals in therapeutic studies such as pig (Evans et al., 1990; Piedrahita et al., 1990; Hochereau-de-Reviers and Perreau, 1993; Gerfen and Wheeler, 1995; Miyoshi et al., 2000; Li et al., 2004a; Kim et al., 2007) and human embryos as well (Heins et al., 2004, Kim et al., 2005). The whole blastocyst culture method is much simpler than other methods described above because it does not need any special chemical reagent or high instrument platform. For this method, the zona pellucida can be removed from embryos by using any suitable method. The zona-free embryos are then plated onto feeder cells. A few days after plating, the attachment of the whole embryo to feeder layer can be seen. The TE cells collapsed and begin to expand, whereas ICM-like cells form a dome shape surrounded by differentiated TE cells. When the ICM clump looks big enough, a finely pulled pipette is used to picked off the ICM clump and transferred it to fresh feeder cells. The differentiated TE cells are left in the plate or discarded after ICM clumps are picked off. This simple procedure, however, runs a much greater risk of TE cell overgrowth than the other methods, because the entire TE cells are cultured along with the ICM clump for several days. The ICM cells are often covered with differentiated TE cells resulting in unclear observation of ES cell morphologies. The ICM might not grow properly, might degenerate or eventually differentiate (Li et al., 2003). To avoid these problem, partial blastocyst culture is used in some cases instead of whole blastocyst culture. The partial blastocyst culture protocol is similar to whole blastocyst culture, except part of the TE cells will be cut off by a mechanical technique such as fine needles or glass pipette. To do so, parts of the TE cells will be removed resulting in significant reduction of the risk of TE cells overgrowth, which tends to inhibit the growth of ICM cells (Lee et al., 2003; Kim et al., 2005). The zona intact blastocyst culture is another method of choice to establish ES cells. Kim and colleagues (2007) cultured zona-intact pig blastocyst stage embryos and compared them with zona-free blastocysts and ICMs (derived from immunosurgery). They found that the number of attached blastocysts of zona intact embryo
Lorthongpanich C. et al./ Thai J Vet Med. 2011. 41(1): 11-22. (27.8%; 15/54) was lower than zona-free embryo (42.4%; 36/85) and significantly lower than those from immunosurgery (68.5%; 87/127). The significant differences were also observed in the rates of primary colony formation of ICMs derived by immunosurgery (36.8%; 32/87), which significantly increased in the group of zona-free (19.4%; 7/36) while no primary colony formed in the zona-intact group (0%; 0/15). Even though, zona-intact embryo culture is one of the pathogen free techniques, it is not often used because this technique has resulted in lower success rates of ES cell establishment (Kim et al., 2007). This might be because of the difficulty of the hatching process, which is a problem often found in in vitro cultured embryos. 1.5 Laser Dissection Laser applications have been used in assisted reproductive technology (ART) for several years including assisted hatching (Obruca et al., 1994), embryo or polar body biopsy (Veiga et al., 1997; Montag et al., 2000), sperm immobilization (Montag et al., 2000), and ICSI (Rienzi et al., 2001), all of which resulted in significantly high fertilization and pregnancy rates of in vitro produced embryos (Obruca et al., 1994; Tanaka et al., 2006). Laser assisted ICM isolation was first used for mouse ICM cells isolation in 2006 (Cortes et al., 2006; Tanaka et al., 2006). Their results demonstrated that the laser dissection method had no influence on ICM attachment and ES cells derivation when compared with the zona-free whole blastocyst culture and ICMs derived immunosurgery, but was significantly higher than zona-intact blastocyst culture. In 2008, Turetsky and colleagues (2008) used the erbium-yttrium-aluminium-garnet (Er: YAG) laser to isolate ICM cells from human embryos (Turetsky et al., 2008). Eight ICMs were isolated from nine hatched blastocysts, which gave rise to three hES cell lines (37.5%; 3/8). This efficiency is similar to the reported after isolation of the ICM by immunosurgery (Thomson et al., 1998; Cowan et al., 2004; Ludwig et al., 2006; Mateizel et al., 2006). In general, laser assisted ICM isolation is performed as follows: The zona-intact blastocysts are fixed by two holding pipettes with the ICM positioned at either nine o'clock (Tanaka et al., 2006) or three o’clock (Turetsky et al., 2008). In mouse embryos, approximately 10 infrared laser pulses at 300 mW x 1 ms (ZILOS-tk™, Hamilton Thorne Research, Beverly, MA USA) are fired to split the blastocyst into two unequal portions the smaller consisting of ICM, the larger consisting exclusively of trophoblast; whereas in human embryos, 20-30 infrared laser pulses at 200 mW x 0.5 ms are used. However, special attention must be paid to direct the laser beam far enough from the ICM to prevent heating and damage of the ICM. Additionally, during the laser dissection experiment, there is a 40% possibility that some blastocysts will not undergo successful ICM isolation, because the TE cells collapse during the laser drill (Turetsky et al., 2008). 1.6 Digestive Method The digestive method is also possible for ICM cells isolation (Li et al., 2003; 2004b). Currently,
15 two reagents have been used for TE cells digestion, enzymatic (Trypsin/EDTA) and Acidic Tyrode’s solution. The enzymatic method has been used with pig embryos to compare the efficiency of isolation among whole blastocyst culture, immunosurgery and the enzymatic method. For the digestive method, blastocyst stage embryos must be treated with pronase or acidic Tyrode’s solution to remove the zona pellucida. Then, the zona-free embryos are submerged into a microdrop of 0.25% trypsin-0.04% EDTA solution for several minutes. During the treatment, embryos are observed under stereomicroscope for the dispersion of TE cells. When the TE begins to disperse, the blastocysts are transferred to another drop with culture medium, and ICM cells are isolated from the dispersed TE cells by the aid of two needles and a pulled mouth pipette. However, it should be noted that the whole embryo culture method has a lower attachment than ICM isolated by enzymatic method (Li et al., 2003). Eightyfive percent of the ICM cells are successfully recovered from blastocyst treated by the enzymatic method as opposed to 40% from immunosurgery (Li et al., 2004b). The ICMs obtained by the enzymatic method prove to be pluripotent and can be differentiated into other types of cells. Acidic Tyrode's Solution is a chemical defined solution, which is widely used to remove all or some zona pellucida from embryos (Cowan et al., 2004; Ellerstrom et al., 2006). It is an effective medium and very quickly removes zona pellucida within a few seconds. Because of the chemically defined solution, acidic Tyrode’s solution is much more suitable for a pathogen-free system than pronase which is a product of bacteria. Ellerstrom and colleagues (2006) used acidic Tyrode’s solution for ICM isolation from human embryos. The blastocysts were incubated in acidic Tyrode’s solution and carefully observed until zona pellucida and TE cells were eliminated. They suggested that 30-40 seconds were the optimum time to remove both zona pellucida and TE cells. However, this treatment could not be used to completely destroy TE cells without damaging the ICM cells. Therefore, the initial outgrowth from the treated blastocysts composed of a heterogenous cells population. However, areas of morphologically distinct ES cells appeared which could be picked off and transferred to fresh plates. Homogenously expressed Oct4 and morphologically resembled undifferentiated hESCs, with a small cytoplasm-tonucleus ratio, could be found at around the fifth passage (Ellerstrom et al., 2006).
2. ES Cell Lines Derivation from Single Blastomeres Since 1981, ES cells have been successfully established from several species including mice (Evan and Kaufman, 1981; Wakayama et al., 2007), monkeys (Thomson et al., 1995; Suemori et al., 2001), and humans (Baharvand et al., 2006; Heins et al., 2006). Although most of the currently available ES cell lines were derived from the ICM cells of a blastocyst stage embryo, it was noted that only a small number of blastomeres from eight-cell (Delhaise et al. 1996) and
16 16-cell (Eistetter, 1989) mouse embryos were viable for deriving ES cells. However, those resulted ES cells can still be considered established by an embryo destruction technique. Therefore, they are still faced with ethical issues, and equally important, immunecompatibility can not be expected from ES cell lines derived from ICM of blastocyst stage embryos. Chung and colleagues (2006) reported an alternative method of establishing ES cell lines using a technique of single blastomere biopsy, which is similar to the techniques used in pre-implantation genetic diagnosis (PGD) in IVF clinics. A single biopsied blastomere will not interfere with the developmental potential of the biopsied embryos (Chung et al., 2006). In PGD, the most common technique involves removing a single blastomere from the eight-cell embryo. The blastomere is analyzed to detect genetic abnormalities; the resultant seven-cell embryos that have no abnormalities are then transferred to the mother’s uterus for implantation and pregnancy. After the novel report by Chung and colleagues (2006), there were several further attempts in mice, monkey and humans that have been reported (Klimanskaya et al., 2006; Wakayama et al., 2007; Teramura et al., 2007; Chung et al., 2008; Lorthongpanich et al., 2008a,b; Narita et al., 2008). However, there are also some new techniques discovered by those people besides culturing the single blastomere as described by Chung and colleagues (2006). The following summarizes techniques relating to ES cells establishment from a single blastomere. The techniques will be grouped into three categories including (1) single blastomere co-cultured with ES supporting cells, (2) whole blastocyst derived from single blastomere outgrowth and (3) immunosurgery of blastocyst derived from single blastomere. The first technique, co-culturing a single blastomere with ES supporting cells, was reported in mouse (Chung et al., 2006) and human embryos (Klimanskaya et al., 2006; Chung et al., 2008). Single blastomeres were biopsied from eight-cell stage embryos and each separated blastomere was aggregated with a small clump (around 100 cells) of GFP-positive ES cells (ES supporting cells) in a 300µm depression created by pressing a needle into the bottom of a plastic tissue culture plate. After 24-48 hours of incubation in ES cell culture medium supplemented with 2,000 U/ml mouse leukemia inhibitory factor (LIF) and 50mM MEK1 inhibitor, a GFP negative bud was observed on the side of ES supporting cells. Then, the aggregates were transferred to feeder cells and ES culture medium until the GFP negative cells were large enough to subculture. The GFP-negative cells were separated from ES supporting cells by microcapillary under fluorescent microscope. The GFP-negative cells were then expanded and tested for ES cell markers. Not only mouse ES cell lines, but also extraembryonic (TE) stem cell lines could be established by single blastomere culture with ES supporting cells (Chung et al., 2006). The second method, whole blastocysts derived from single blastomere outgrowth, was reported a year later by Wakayama and colleagues (2007). The biopsied blastomeres were individually
Lorthongpanich C. et al. / Thai J Vet Med. 2011. 41(1): 11-22. cultured in 96 well plates precoated with feeder cells in ES culture medium with 20% Knockout Serum Replacement (KSR) and 0.1 mg/ml adrenocorticotropic hormone (ACTH; fragments 1–24) instead of fetal calf serum (FCS). During the time that the blastomeres were in culture, they could divide and develop into blastocysts. After 10 days or more, proliferation outgrowths were dissociated and replated to expand until stable ES cell lines grew out. Using this technique, they could produce several ES cell lines from single blastomeres derived from twocell (50-69%), early four-cell (28-40%), late four-cell (22%), and eight-cell (14-16%) stage embryos (Wakayama et al., 2007). The last method is immunosurgery, where the blastocyst is derived from a single blastomere. Since the single blastomere derived blastocyst contains a small amount of ICM cells (Lorthongpanich et al., 2008a), there are not many attempts to use immunosurgery with a single blastomere derived embryo. However, there was a paper describing the successful use of immunosurgery with blastocysts derived from single blastomeres of two-cell stage embryos that contained prominent ICM cells (Teramura et al., 2007).
3. Current Situations of ES cell Established from Single Blastomere Deriving embryonic stem cells from single blastomeres is an effective technique to overcome the ethical concerns for establishing ES cells, especially hES cells, because this is a non-destructive embryo technique. However, as it has only been in use for a few years, there still have many profound problems that need to be studied. Some of the more interesting problems are why, in the more advanced embryonic stage, the success rate of ES cell establishment is less; and how can the success rate of ES cell establishment be improved. In addressing those problems, we may be able to look at two major factors, which are blastomere fate determination and the culture system. We know that during the pre-implantation embryo development, two distinct segregated cell lineages are established late in the morula stage. The outer cells of the embryo will become TE cells and the inside cells will become ICM cells. Lineage segregation has also been studied in early embryos for almost 50 years. Currently, there are two active models that attempt to explain the segregation fate in early embryos. One posits that lineage is not predetermined until late morula stage, whereas another suggests that fate is already determined very early in the embryo development. Up to now, the early embryonic sister blastomeres differentiation competence is still one of the fundamental questions that has not been fully explained (Tarkowski et al., 1959; Takowski and Wroblewska, 1967; Rossant, 1976; Tsunoda and McLaren, 1983; Papaioannou et al., 1989; Chan et al., 2000; Piotrowska et al., 2001; Piotrowska-Nitsche et al., 2005). There have been several attempts at trying to produce a full set of offspring after transfering isolated blastomeres derived embryos and splitting embryos to surrogates. Unfortunately, not all blastomeres were able to develop to term (Rossant,
Lorthongpanich C. et al./ Thai J Vet Med. 2011. 41(1): 11-22. 1976; Tarkowski et al., 1959; Tsunoda and McLaren, 1983; Papaioannou et al., 1989; Heyman et al., 1998; Chan et al., 2000; Mitalipov et al., 2002; Schramm and Paprocki, 2004). Even though the production of monozygotic twins by separating two-cell embryos has been achieved in some species (Willadsen, 1979; Willadsen et al., 1981; Willadsen et al., 1981; Ozil et al., 1982; Allen and Rashen, 1984; Tsunoda et al., 1984; Matsumoto et al., 1989; Papaioannou et al, 1989) only one report showed the pluripotency of four-cell blastomeres which produced quadruplets (four identical calves) (Johnson et al., 1995). It has also been found that although each two-cell stage blastomere gives rise to both the ICM and the TE lineages, one cell tends to contribute more to the embryonic part of the blastocyst, and the other cell contributes to the abembryonic part (Gardner, 2001; Piotrowska-Nitsche et al., 2001; Fujimori et al., 2003; Piotrowska-Nitsche and Zernicka-Goetz, 2005). Additionally, at the fourcell stage, Piotrowska-Nitsche and colleagues (2005) demonstrated that each blastomere had predictable fates. The allocation bias of a two- and four-cell blastomere progeny suggests the differential developmental competence of the sister blastomeres and their lineage fate. As reported by Chung and colleagues (2006), the blastomeres were biopsied from eight-cell stage embryos and were co-cultured with ES supporting cells. They could produce both ES cell lines and extraembryonic (trophoblastic) stem cells lines. These also suggest the bias of the blastomeres of eight-cell stage embryos. However, several reports have demonstrated the non-developmental bias in early stage blastomeres. Therefore, whether or not the lineage fate is committed at an early embryonic stage remains unclear. Another factor that might affect the success rate of ES cell establishment from a single blastomere is the culture system. This technique was originally developed by the culture of a single blastomere in conventional mouse ES medium supplemented with some protein inhibitor reagents and/or co-cultured with ES supporting cells. MAPK
17 family is the common pathway involved in stem cell research because of its roles in regulating proliferation, differentiation and apoptosis (Binetruy et al., 2007). Some of the MAPK inhibitors have been used for the derivation of ES cells from single blastomeres such as MAP2K (I) and MAPK14 (I) (Chung et al., 2006; Lorthongpanich et al., 2008b). Although single blastomeres derived ES cell lines have been successfully established from MAP2K (I) supplementation and co-culturing with ES supporting cells, the role of MAP2K (I) and ES supporting cells have not yet been determined (Chung et al., 2006). MAPK14 (I) has been reported to inhibit TE cells differentiation of mouse morula stage embryo. This suggests the potential of enhancing ICM development by suppressing TE cells (Maekawa et al., 2005). MAPK14 (I) has been supplemented into culture medium for single blastomere culture and ES cell derivation. The results demonstrated that MAPK14 (I) could inhibit the development of blastomeres derived embryos and also inhibit TE cell differentiation which is similar to its action on an intact embryo. However, MAPK14 (I) could not enhance ICM development in single blastomeres derived embryos, but instead inhibited ICM formation (Lorthongpanich et al., 2008b). Adrenocorticotropic (ACTH) is a peptide hormone produced from the pituitary gland responsible for an important role in early development. ACTH has been found to enhance ES cell proliferation from ICM derived ES cell lines (Ogawa et al., 2004). Recent studies have further confirmed the role of ACTH in supporting the derivation of ES cells from single blastomeres (Wakayama et al., 2007; Lorthongpanich et al., 2008b; Narita et al., 2008). However, supplementation with ACTH is not enough to ensure that every blastomere derived from the same embryo become ES cells. The result of ACTH studies also suggested that blastomeres derived from higher stage embryos had lower success rates than those blastomeres derived from lower stage embryos (Wakayama et al., 2007).
Table 1 Comparison of each method for ES cells establishment from ICMs of blastocyst stage embryos Method Microsurgery
Mechanism Use micro blade or fine needles to cut TE cells
Prominent point Simple method
Specification of antibody against antigen on TE cells surface Osmotic pressure change in TE cells causes cell lyses Remove ZP or partially cut TE cells and ready to plate onto feeder cells Laser drill to destroy TE cells under
Complete destruction of TE cells
Calcium Ionophore (A23187) Whole or partial blastocyst culture Laser dissection
Use Trypsin/EDTA or Acidic Tyrode’s solution to digest TE cells
Simple method Most simple method, Pathogen-free Pathogen-free
Weak point Crude method, Takes time, Difficult to make ICM with clear TE cells, ICM might have losses during processing, Depending on the operator technical’s skills Pathogen-contamination risk, ICM might have losses during processing Uncontrollable of various effects of A23187 TE cells overgrowth
Need Laser set and operator’s micromanipulator skill, Takes time, Heat from laser beam Pathogen-contamination risk (Pronase), ICMs might be hurt from digestion process
Lorthongpanich C. et al. / Thai J Vet Med. 2011. 41(1): 11-22.
Conclusion ICM isolation is an early step that determines whether ES cells are considered pathogen free. When generating ES cell lines, there are some criteria that need to be considered before choosing one of the methods for ICM cells isolation. First, we need to consider whether the ES cells need to be xeno-free, then, what quality of embryo is needed, and finally, if your laboratory has a manipulator and a skillful operator. As shown in Figure 1, when using the conventional method, one must first decide whether the ES cells need to be xeno-free. If they do, then the embryos need to be separated into (at least) two groups, good and poor quality. The poor quality embryos with unclear and small ICM cells should use zona-free whole blastocyst culture otherwise the tiny ICM cells may be lost during processing in subsequent complicated procedures. For good blastocysts, there are many options depending on your manipulator’s skills. For skillful people, laser dissection or mechanical isolation with micro blade or with ultra fine pulled glass needles could be a method of choice. On the other hand, partially cutting off the
TE cells with fine needles, calcium ionophore (A23187) or Trypsin digestion methods can be used by a person with lesser skills. However, if the ES cells do not need to be xeno-free, you can also use immunosurgery for ICM cells isolation from a good blastocyst. Even though there are several methods that can be used at present, it is unclear which approach is more efficient. Therefore, the most suitable method needs to be considered if it fits the skills of the operator (Table 1 and Table 2). A novel method, single blastomere derived ES cells, opens a new era for ES cell establishment with less ethical problems. However, since this technique is new, there are many questions such as blastomeres fate, efficiency of each blastomeres from different embryonic stages to derive ES cells and a suitable culture system that need to be clarified. At present, there are several laboratories attempting to overcome these obstacles and developing techniques to derive ES cells that can reach maturity from single blastomeres (Green et al., 2009; Roberts et al., 2010).
Figure 1 Diagram of ICM isolation method consideration
Table 2 Comparison of each method for ES cells establishment from single blastomeres co-culture and non co-culture with ES supporting cells Method Co-culture
Mechanism Single blastomere co-culturing with ES supporting cells for several days
Prominent point Increase chance success
Single blastomeres are cultured individually to blastocysts and outgrowths
Acknowledgement This study was supported by The Royal Golden Jubilee Ph.D. Program of Thailand Research Fund and Suranaree University of Technology. The authors would like to thank Leslee Sinclair for critical review.
Weak point Unclear mechanism of ES supporting cells, Contamination risk from supporting cells, Unequal competence of each blastomere Unequal competence of each blastomere
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Fas Ligand in Swamp Buffalo Oviduct during Follicular and Luteal Phases Paisan Tienthai1* Pongpisut Chivacharern1 Kriengyot Sajjarengpong1 Pornchalit Assavacheep2
Abstract Fas ligand (FasL) and its receptor (Fas) are tumor necrosis factor (TNF) members which are involved in the immune privileged organs, such as the cornea, testis and placenta, by triggering apoptosis in various cell types. The appearance of a Fas-FasL system might specify the immune privileged status of the buffalo oviduct where spermatozoa avoid eradication by female immune cells. The objective of this study was to scrutinize the FasL and Fas proteins by immunohistochemistry, FasL mRNA by RT-PCR and the apoptotic degrees by TUNEL analysis in the uterotubal junction (UTJ), isthmus, ampulla and infundibulum of swamp buffalo oviducts at follicular (n=5) and midluteal (n=5) phases. The Fas localization was scattered along the lining epithelium of all oviductal portions at both estrous cycle stages, whereas the intensity of FasL immunostaining was more noticeable in the epithelial cells of UTJ and isthmus at follicular phase and showed significant differences (p<0.05) compared to other segments and to the mid-luteal phase. FasL mRNA expression was detected in the epithelial cells of all oviductal segments and the intense of expression in UTJ and isthmus at follicular phase was greater than similar regions at mid-luteal phase and other segments. However, the TUNEL-apoptotic cells were rarely detected in all oviductal segments of swamp buffalo at both phases. The present results indicate that the appearance of Fas and FasL in the UTJ and isthmus which are the site of sperm reservoir, at follicular phase can be involved in Fas-FasL system that mediate the survival of spermatozoa and supports the immune privilege status in the swamp buffalo oviduct. Keywords: estrous cycle, Fas-FasL, oviduct, sperm reservoir, swamp buffalo 1Department
of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand 10330. of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand 10330. Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 23-31.
Paisan T. et al. / Thai J Vet Med. 2011. 41(1): 23-31.
Introduction Sperm storage and capacitation, ovum pickup, fertilization including early embryonic growth are critical situations taking place in the cattle oviduct (Hunter and Wilmut, 1984; Lefebvre et al., 1995). The mucosal surface in the female reproductive tract is protected by innate and adaptive immune defense regulations (Wira et al., 2005). The innate defense mechanism is composed of the tight junction of epithelial cells, antimicrobial secretions and phagocytic immune cells, whereas the intraepithelial lymphocytes primarily serve as the adaptive mechanism (Mowat, 2003). After insemination, large amounts of spermatozoa that contain foreign proteins are demolished by neutrophil phagocytosis and other immune cells responses within the uterine horns (Katila, 2001; Kaeoket et al., 2003; Matthijs et al., 2003). After rapid transport in cattle, hundreds of
spermatozoa are arrested in the uterotubal junction (UTJ) and adjoining isthmus, the site defined as the sperm reservoir, where they can survive the microenvironment for several hours until release when the ovulation occurs (Hunter and Wilmut, 1984). Unlike the uterine horns, these spermatozoa are able to hide away the mucosal immune response of the oviduct. In inseminated pigs at pre-ovulation, RodriguezMartinez et al. (1990) indicated that the neutrophils were absent from the UTJ and the caudal isthmus was irrelevant to the appearance of boar spermatozoa. Furthermore, the intraepithelial immune cell numbers in non-inseminated swamp buffalo oviduct were very low at follicular phase in UTJ compared to the other oviductal segments (Tienthai et al., 2008). According to these observations, some regions of the buffalo oviduct, particularly in the UTJ and adjacent isthmus, might function as an immune privileged organ corresponding to the testis (Lee et al., 1997; Koji et al., 2001), the placenta (Hammer et al., 1999; Kauma et al.,
Paisan T. et al./ Thai J Vet Med. 2011. 41(1): 21-29. 1999), or the cornea in the eyes (Stuart et al., 1997) where cells expressing foreign proteins escape rejection. Immune privileged organs and tissues generally have a specific regulation to allow the foreign proteins to survive the immune responses and the Fas-Fas ligand (FasL) system has been implicated as an essential mediator for this regulation (Griffith et al., 1995). FasL is a type II transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily and the Fas receptor is a type I transmembrane protein with a death domain in its cytoplasmic region that stimulates an apoptotic signal when binding to FasL (Suda and Nagata, 1994). It is believed that the localization or expression of FasL accords to the immune privilege by inducing Fasmediated apoptosis in lymphocytes that distribute into FasL-bearing tissue (Nagata, 1997). For instance, the remarkable appearance of FasL in the glandular epithelium and the decidual cells in the placenta and the capability of trophoblasts to induce FasLdependent apoptosis, indicate the role of Fas-FasL system in maternal immune tolerance toward the fetus (Mor et al., 1998; Kauma et al., 1999). The detection of Fas-FasL interaction in the swamp buffalo oviduct can explain the local immune tolerance that prevents the spermatozoa from cytotoxic T lymphocytes. Therefore, the present study was performed to investigate Fas-FasL immunolocalization, FasL mRNA expression and apoptotic analysis in all segments of the swamp buffalo oviduct during the follicular and mid-luteal phases.
Materials and Methods Animals and tissue collection: Female swamp buffalo (n=20) of various ages (2-8 years) were slaughtered at a local abattoir and their genital tracts were immediately collected and reserved in a cool container (~4oC) for at least 30-45 min until being processed in the laboratory. The reproductive organs were observed and the ovarian characteristics were determined by the morphological appearance of the corpus luteum (Ali et al., 2003), i.e. follicular (n=10) and mid-luteal (n=10) phases. The swamp buffalo oviducts were divided into uterotubal junction (UTJ), isthmus, ampulla and infundibulum. The samples for RNA extraction (the follicular phase, n=5 and the luteal phase, n=5) were promptly deep-frozen in liquid nitrogen (LN2), whereas the specimens for immunohistochemistry and TUNEL assay (follicular phase, n=5 and luteal phase, n=5) were immersed in 4% paraformaldehyde at 4oC until being embedded in paraffin. Tissue blocks were cut at 4 µm thickness and serial sections were mounted on Superfrost plus slides (Menzel-Graser, Frieburg, Germany) for Fas, FasL immunohistochemical and TUNEL techniques. Immunohistochemical procedure for Fas and FasL: After deparaffinization and rehydration, tissue sections were quenched with 3% hydrogen peroxide (H2O2) in methanol and washed in phosphate buffer saline (PBS, pH 7.4). For the primary antibody, mouse anti-human Fas monoclonal antibody (B-10, Santa
25 Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-rat FasL polyclonal antibody (N-20, Santa Cruz) were used. Briefly, sections were blocked with 10% normal horse serum (for FasL) or normal goat serum (for Fas), prior to incubation overnight at 4oC in a humidity chamber with a 1:100 dilution of Fas antibody or a 1:150 dilution of FasL antibody. After rinsing with PBS, bound antibody was detected by the avidin-biotin-peroxidase method, using reagents of the Vectastain ABC kit (Vectastain ABC-Elite, Vector Laboratories, Burlingame, CA, USA). Biotinylated horse anti-mouse IgG (Vector Laboratories) at a dilution of 1:500 was used for Fas, whereas biotinylated goat anti-rabbit IgG (Vector Laboratories) at a dilution of 1:400 was used for FasL. For both Fas and FasL antigens, bound peroxidase were reacted with 3,3’-diaminobenzidine substrate (DAB kit, Vector Laboratories) with H2O2 to give a brown reaction product. All slides were counterstained with hematoxylin and mounted in glycerin-gelatin. As negative controls, adjacent sections were reacted in parallel with a substitution of mouse IgG (dilution 1:100, DAKO, Glostrup, Denmark) for primary antibodies. Ovarian sections prepared from adult mice were used as positive controls. The stained slides were then investigated under light microscope (BX50, Olympus, Tokyo, Japan) equipped with a digital camera Micropublisher 5.0 (Qimage, Surrey, BC, Canada) and software program (Image-Pro Plus 6.0 (Media Cybernatics Inc., Bethesda, MD, USA). Examination of Fas and FasL positive immunostaining in the surface epithelium of all oviductal segments was carried out by blind preparation. The intensity of positive staining was categorized into four different scores as follows: no staining, 0; weak, 1; moderate, 2 and strong, 3. Extraction of total RNA and reverse transcriptionpolymerase chain reaction (RT-PCR): The tubal epithelium from the UTJ, isthmus, ampulla and infundibulum of swamp buffalo oviduct was scraped using the blunt side of a scalpel blade. Total RNA was isolated from the epithelial cells by use of the RNeasy mini kit (QIAGEN GmbH, Hilden, Germany). Synthesis of cDNA and PCR was performed using Transcriptor One-Step RT-PCR kit (Roche, Mannheim, Germany). Amplification conditions were as follows: denaturation 94oC for 30 sec, annealing 55oC for 30 sec, extension 72oC for 30 sec, for 44 cycles. The primers used were designed for bovine FasL (SigmaGenosys Ltd., Pampisford Cambridgeshire, UK). Sense and anti-sense specific primers were as follows: 5’-TATTCCAAAGTATACTTCCGGGGT CA-3’ and anti-sense 5’-ACTGCC CCCAGGTAGCTGCTG-3’ (Genebank accession number U95844). Apoptotic analysis by TUNEL assay: After being deparaffinized and rehydrated, the tissue sections were pretreated with proteinase K for 15 min at room temperature (RT). The Terminal Deoxynucleotidyl Transferase-mediated dUTP nick-end labeling (TUNEL) assay to evaluate apoptotic cells using an ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., CA. USA) was performed according to the manufacturer’s
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instructions. In brief, endogenous peroxidase activity was reduced with 3% H2O2 in PBS for 5 min at RT. After rinsing, the slides were treated with equilibration buffer for 5 min at RT and then incubated with TdT enzyme for 1 hr at 37oC using a parafilm cover. A stop buffer was added for 10 min at RT, followed by washing in PBS and incubating with an anti-digoxigenin conjugate for 30 min at RT. To visualize the labeled 3’-OH ends of DNA fragments, 3,3’-diaminobenzidine substrate (DAB kit, Vector laboratories) with H2O2 was applied for 1-3 min. The slides were then rinsed in distilled water, counterstained with Mayer’s hematoxylin and mounted with glycerine gelatin. Negative control sections were incubated with PBS instead of TdT working enzyme whereas canine lymph nodes with lymphoma were used as a positive control. An ocular micrometer with 25 squares corresponding to 15,625 µm2 at magnification 400x was used for counting the TUNEL-positive cells along the lining epithelium. For each oviductal segment, 20 microscopic areas were randomly selected for evaluation. Statistical analyses: Data was handled and analyzed using the SAS statistical package (version 9.0, SAS Institute Inc., Cary, NC, USA). The intensity score of Fas-FasL immunostaining and the number of TUNELpositive cells were compared between the oviductal segments and estrous cycle stages using one-way factorial ANOVA. Differences between means were determined by a student t-test, p values <0.05 were considered statistically significant.
Figure 2 Fas immunolocalization in the epithelium of the UTJ (a, b), isthmus (c, d), ampulla (e, f), and infundibulum (g, h) of the swamp buffalo oviducts at the follicular (a, c, e, g) and mid-luteal (b, d, f, h) phases of the estrous cycle.
Results Immunolocalization of Fas proteins: In control sections, Fas-positive immunolocalization appeared in the cytoplasm of follicular granulosa cells surrounding the oocyte in the mouse ovary (Fig. 1a),
Figure 1 Control sections for Fas immunohistochemistry. Mouse ovarian section was used as the positive control (a); Omission of the primary antibody and substitution by a mouse IgG on UTJ section was used as the negative control (b).
immunolabeling was present in the cytoplasm of epithelial cells, especially in the apical part of cells, and the staining was dispersed along the lining epithelium at both phases of the estrous cycle (Fig. 2ah). The moderate to strong staining of Fas-positive labeling was varied along the oviduct, but no obvious differences in the intensity scoring could be observed among portions (Fig. 3A) or between phases of the estrous cycle (Fig. 3B).
Figure 3 Intensity scores of Fas immunostaining in the lining epithelium of the UTJ, isthmus, ampulla and infundibulum of the swamp buffalo oviduct during the follicular and mid-luteal phases of the estrous cycle. (A) Staining intensity was compared between segments at the same phase and (B) was compared between phases. Values are depicted as mean±SEM with different labels (a, b) being significantly different (p<0.05).
Paisan T. et al./ Thai J Vet Med. 2011. 41(1): 21-29.
Figure 4 Control sections for FasL immunohisto chemistry. Mouse ovarian section was used as positive control (a); Omission of the primary antibody and replacement with a mouse IgG on UTJ section was used as the negative control (b).
27 UTJ and isthmus, at the follicular phase (Fig. 5a, c, e, g) compared to the mid-luteal phase (Fig. 5b, d, f, h). At the follicular phase, manual intensity scoring confirmed that the FasL labeling in the UTJ and isthmus was significantly higher (p<0.05) than the ampulla and infundibulum (Fig. 6A). In addition, the FasL intensity in the UTJ and isthmus at the follicular phase was significantly greater (p<0.05) than the same regions at the luteal phase (Fig. 6B).
Immunolocalization of FasL proteins: In the control sections, FasL-positive immunolocalization was also clearly found in the cytoplasm of follicular granulosa cells (Fig. 4a), whereas there was no immunostaining on the negative controls (Fig. 4b). Similarly to the Fas proteins, FasL-positive staining was shown in the cytoplasm of epithelial cells, which could not identify the cell types, along the oviductal segments (Fig. 5). However, the intense immunolocalization of FasL protein was conspicuously present in the epithelial cells of the swamp buffalo oviduct, especially in the
Figure 6 Intensity scores of FasL immunostaining in the lining epithelium of the UTJ, isthmus, ampulla and infundibulum of the swamp buffalo oviduct during the follicular and mid-luteal phases of the estrous cycle. (A) Staining intensity was compared between segments at the same phase and (B) was compared between phases. Values are depicted as mean±SEM with different labels (a, b) being significantly different (p<0.05).
FasL mRNA expression: An expected 168 bp band was investigated in all samples of the swamp buffalo oviduct at both estrous cycle stages (Fig. 7). At follicular phase, the FasL mRNA expression was more abundant in the UTJ and isthmus than in the ampulla and infundibulum as well as the oviductal segments at luteal phase.
Figure 5 FasL immunolocalization in the epithelium of the UTJ (a, b), isthmus (c, d), ampulla (e, f), and infundibulum (g, h) of the swamp buffalo oviducts at the follicular (a, c, e, g) and mid-luteal (b, d, f, h) phases of the estrous cycle.
Figure 7 FasL mRNA expressions in the epithelium of the uterotubal junction (UTJ), isthmus (UST), ampulla (AMP) and infundibulum (INF) of the swamp buffalo oviduct during the follicular (F) and midluteal (L) phases of the estrous cycle.
Table 1 Number of TUNEL-positive apoptotic cells (mean±SD/ area of ocular micrometer 15625 µm2) in different segments of swamp buffalo oviduct at follicular and mid-luteal phases Oviductal segments/ Estrous cycle Follicular Mid-luteal
28 Detection of apoptosis: The lymph nodes (lymphoma) which were used as positive controls for TUNEL assay demonstrated numerous positive apoptotic cells or apoptotic bodies (brown staining) throughout the tissue sections (Fig. 8a), whereas none of the cells in the negative control (ampulla section) were labeled (Fig. 8b). In the swamp buffalo oviduct, however, very few apoptotic cells were detected in any segments at both phases of the estrous cycle (Fig. 9) and the number of TUNEL-positive cells was not significantly different (p>0.05) between segments or estrous phases (Table 1).
Figure 8 Control sections for TUNEL assay. Canine lymph node (lymphoma) section (a) showed TUNELpositive brown staining (arrows); UTJ section replacement with mouse IgG was used as the negative control (b).
Figure 9 Detection of apoptotic cells (arrows) by TUNEL assay in the epithelium of the UTJ (a, b), isthmus (c, d), ampulla (e, f), and infundibulum (g, h) of the swamp buffalo oviducts at the follicular (a, c, e, g) and mid-luteal (b, d, f, h) phases of estrous cycle.
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Discussion The present study confirmed that protein localization and mRNA expression of FasL was found in the swamp buffalo oviductal epithelium, especially in the UTJ and adjacent isthmus (the site of sperm reservoir), at the follicular phase which was the time of sperm storage and fertilization. Contrastly, Fas immunostaining was clearly present along the lining epithelium of all oviductal portions at all estrous cycle stages. Although the apoptotic cells were rarely observed by TUNEL assay, the present investigation suggested that a regulation of Fas-FasL system occurred within the sperm reservoir of the swamp buffalo oviduct. It is known that the main functions of the cattle oviduct in relation to critical events, i.e. sperm transport, oocyte collection, fertilization and initial embryonic growth, take place in 2-3 days subsequent to the beginning of the estrus period (Lefebvre et al., 1995; Suarez, 2002). Several studies found that spermatozoa contained proteins that were considered to be a foreign to the female immune system and the presence of spermatozoa and seminal plasma in the uterine horns after mating or artificial insemination induced the distribution of polymorphonuclear leukocytes and other immune cells from the subepithelial connective tissue layer of the endometrium to eliminate them (Katila, 2001; Kaeoket et al., 2003; Matthijs et al., 2003). Hunter et al. (1991) found that the caudal isthmus and UTJ functioned as the sperm reservoir in cattle where the spermatozoa remained viable before fertilization. Altogether, this specific region, e.g. in pigs, demonstrates a minimal immune response by leukocytes after insemination (Rodriguez-Martinez et al., 1990) corresponding to the swamp buffalo oviduct that shows a small number of immune cells within UTJ and isthmus epithelium throughout the estrous cycle (Tienthai et al., 2008). One of the essential factors involved in sperm survival in the sperm reservoir is the presence during the pre-ovulatory stage of mucous hyaluronan that contains the immunologically static condition to assist the immersed spermatozoa escape identification by immune cells (Tienthai et al., 2000; Bergqvist et al., 2005a). However, this event does not explain the decrease of the immune cells in the sperm reservoir of various species and recently the presence of Fas-FasL system can be detected in the bovine oviduct (Bergqvist et al., 2005b) which is thought to have a major role in the limiting of the immune cell numbers in the cattle oviduct. We know that immunologically privileged sites are the regions of the body where the immune system does not appear to function (Griffith, 1995) and the Fas-FasL system apoptotic pathway has been demonstrated to play an important role in the immune system by stimulating the activation inducted suicide of T-lymphocytes and other immune cells (Steller, 1995). In the present study, the FasL immunostaining was found along the epithelial lining of the swamp buffalo oviduct corresponding to the bovine oviduct (Bergqvist et al., 2005b), however, our result confirmed that a strong intensity of FasL
Paisan T. et al./ Thai J Vet Med. 2011. 41(1): 21-29. protein was only present in the epithelial lining of the UTJ and adjacent isthmus at the pre-ovulatory phase in relation to the expression of FasL mRNA. Therefore, the site of the sperm reservoir (UTJ and caudal isthmus) in the swamp buffalo oviduct might also acquire this special mechanism to selectively eliminate immune cells and temporarily maintain sperm ability and also the early embryo. Nagata and Golstein (1995) reported that activated T lymphocytes that expressed Fas and FasL were sensitive to Fasinduced apoptosis indicating that the activated T lymphocytes commited suicide or even killed each other. Recently, the Fas-FasL system was also associated with the process in the decline of natural killer (NK) cells by apoptosis (Kusakabe et al., 2005) and the apoptotic signals produced by trophoblastic FasL regulate the immune traffic including maintaining maternal tolerance by preventing the activated T lymphocytes from entering the fetal cellular compartment (Xerri et al., 1997). This data confirms the expressions of Fas and FasL in both activated T cells and NK cells, and there is the possibility that apoptosis induction in the sperm reservoir of swamp buffalo oviduct will occur when these immune cells (Fas-bearing cells) are distributed into the epithelial lining which demonstrates the intense FasL immunostaining. In the present study, Fas-FasL localization in the cell surface of the intraepithelial or subepithelial immune cells in swamp buffalo oviducts was not observed, therefore, cryostat tissue sections instead of paraffin sections would be required to investigate Fas-FasL immunolocalization (Kusakabe et al., 2005) in any further study. In the present study, the localization of FasL protein was shown in the apical part of the oviductal epithelial cells corresponding to earlier studies in mouse (Imarai et al., 2005) and cow (Bergqvist et al., 2005b) oviducts. Importantly, our technique with paraffin sections was unable to identify which types of oviductal epithelial cells reacted to FasL immunostaining. However, the previous studies by immune-electron microscopic analysis in melanoma cells (Adreola et al., 2002) and ocular epithelial cells (McKechnie et al., 2006) reported that FasL proteins were localized as multi-vesicular patterns, therefore, FasL proteins could be bound to the secretory vesicles or granules. These observations assumed that the positive staining of FasL at the apical portions was in the location of secretory granules within the secretory cells of epithelial cells since being carefully noticed in positive FasL immunolabeling in the UTJ (Fig. 5a) and isthmus (Fig. 5c) of swamp buffalo oviducts. Considering the estrous cycle stage, both FasL protein and mRNA in this study obviously appeared during the follicular phase compared to the mid-luteal phase and one possible reason is that the presence of FasL can be regulated by the influence of estrogen. Song et al. (2002) suggested that FasL proteins expression increased in Ishikawa cells and primary cultures of uterine glandular cells treated with estrogen. Furthermore, Sapi et al. (2002) indicated that high levels of estrogen during estrus definitely upregulated the expression of FasL proteins and mRNA intensity in ovarian epithelial cells and ovarian tissues. These
29 observations not only suggest that the abundance of Fas protein localization and mRNA expression are influenced by estrogen during the follicular phase, but also confirms that FasL is biologically active in the swamp buffalo oviduct. One question raised in our study was whether the Fas-FasL system in the swamp buffalo oviduct might trigger apoptosis in the epithelial cells during the estrous cycle as the presence of Fas-FasL was found in the epithelium of all oviductal portions. In cattle oviducts, the epithelial extrusion of secretory cells markedly occurred in the ampulla and infundibulum at the luteal phase (Abe and Oikawa, 1993; Tienthai et al., 2008; 2009) and this phenomenon was found in the present study (Fig. 2f, h and Fig. 5f, h) as well. Several investigators have reported that the cytoplasmic and nuclear extrusions of oviductal epithelium were influenced by high levels of progesterone and were related to apoptotic events (Verhage et al., 1984; 1990). However, Steffl et al. (2008) recently concluded that this epithelial protrusion was a characteristic feature of nonapoptotic cell loss of secretory (non-ciliated) cells in large animals and dogs which were a species with long luteal phase. In our study, a fewer TUNELapoptotic cells were demonstrated in all segments of the swamp buffalo oviduct at both estrous stages similar to the bovine oviduct (Bergqvist et al., 2005b) suggesting that there was no correspondent between the Fas-FasL localization and apoptosis in the swamp buffalo oviductal epithelium. However, we know that the data associated with apoptosis in large domestic animals’ oviducts is very rare and those studies performed only TUNEL assay which can detect late and short stages of apoptosis (Grossmann et al., 1988), while the Fas-FasL system may be expressed at an earlier stage. To prove exact apoptotic events, the specific and sensitive markers of apoptosis which detect cells in early stages, such as the immunohistochemical detection of apoptotic cells using an anti-single-stranded DNA (anti-ssDNA) antibody (Kawarada et al., 1998; Steffl et al., 2008), might be investigated in the cattle oviduct. Based on our results, however, the FasL immunolocalization and mRNA expression by the epithelial cells of sperm reservoir in the swamp buffalo oviduct might act as an immune privileged site and could induce apoptosis in Fas-bearing immune cells by the mechanism of direct cell-cell contact. In conclusion, the present study indicates the appearance of Fas-FasL proteins and FasL mRNA in the epithelial cells of the swamp buffalo oviduct. Importantly, FasL was particularly present in the UTJ and isthmus at the follicular phase supporting the theory that sperm reservoir in the swamp buffalo oviduct serves as an immune privileged site. However, the mechanisms of the Fas-FasL system in relation to the presence of FasL in various types of immune cells and the apoptotic events by other techniques in the swamp buffalo oviduct still require further investigation.
Acknowledgement The authors would like to thank Mr. Silpchai
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pienchop, Mr. Witoon Mabutr and Mrs. Jantima Intarapunya of Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, for their excellent technical assistance. This study was financially supported by Ratchadaphiseksomphot Endowment Fund, 2009, Chulalongkorn University, Thailand.
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The Virulence of Thai Isolated Mycoplasma gallisepticum in Challenged Embryonated Eggs Somsak Pakpinyo* Suwarak Wanaratana Sarawoot Mooljuntee
Abstract This study was to investigate the virulence and pathogenicity of Thai isolated Mycoplasma gallisepticum (MG) inoculated into embryonated eggs and the chance of virulence study in embryonated eggs instead of experimental chickens. One hundred and twenty eight-day-old embryonated eggs were divided into 4 groups as follows. Group 1 consisting of 12 eggs served as sham negative control and was inoculated with 0.1 ml of broth into yolk sac. Group 2 (2.1, 2.2 and 2.3), 3 (3.1, 3.2 and 3.3) and 4 (4.1, 4.2 and 4.3) were inoculated with 0.1 ml of MG strains F, 6/85 and Thai isolated into yolk sacs, respectively. Each subgroup consisted of 12 eggs and the number of microorganisms differed between each subgroup, 108, 106 and 104 CFU/ml, respectively. Early death period (3-6 days post inoculation) and late death period (7 days and later post inoculation) were observed. When the chicks were 7 days old, blood collection was done for serology by SPA and ELISA. Then all of them were necropsied for gross thoracic airsac lesion score and microscopic tracheal lesion score, and the airsacs of all chicks were swabbed for DNA detection by PCR assay. Results revealed that the early and late death periods of all groups ranged from 0-4 and 2-7 eggs, respectively, and the number of survival chicks 0-6 days old and 7 days old were 0-3 and 1-9, respectively. The mean of thoracic airsac lesion score of dead and survival chicks was 0-1.33 without significant difference, however, significant difference was found when group 1 was compared with groups 3.2, 3.3, 4.1 and 4.2 (p<0.05). The mean of tracheal lesion score was 0.81-2.56 without significant difference. The number of positive reactors against SPA and ELISA was 0 and 1, respectively. The number of positive results against PCR assay was ranged 0-4. However, MG DNA of groups 1 and 2.3 was not observed. This study suggested that evaluation of the virulence and pathogenicity of Thai isolated MG could cause a lesion of thoracic airsac, and embryonated eggs could be used instead of experimental chickens in virulence study. Keywords: antibody, embryonated eggs, Mycoplasma gallisepticum, PCR, thoracic airsac, tracheal lesion score Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330 Thailand. Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 33-38.
Pakpinyo S. et al. / Thai J Vet Med. 2011. 41(1): 33-38.
บทคัดย่อ ความรุนแรงของเชื้อ มัยโคพลาสมา กัลลิเซพติกุม สายพันธุท์ ี่แยกได้ในประเทศไทยโดยใช้ไข่ไก่ฟัก สมศักดิ์ ภัคภิญโญ * สุวรักษ์ วรรณรัตน์ สราวุธ มูลจันที การศึกษาครั้งนี้มีวัตถุประสงค์เพื่อศึกษาความรุนแรงและพยาธิสภาพของเชื้อ มัยโคพลาสมา กัลลิเซพติกุม (เอ็มจี) สายพันธุ์ที่แยก ได้ในประเทศไทยในไข่ไก่ฟักและความเป็นไปได้ของการนําไข่ไก่ฟัก มาศึกษาความรุนแรงของการได้รับเชื้อเอ็มจีแทนตัวไก่ทดลอง ทําการแบ่ง ไข่ไก่ฟักอายุ 8 วัน จํานวน 120 ฟอง เป็น 4 กลุ่ม ดังนี้ กลุ่ม 1 กลุ่มควบคุมลบ (ฉีดอาหารเลี้ยงเชื้อเข้าไปที่ถุงไข่แดง 0.1 มล.) จํานวน 12 ฟอง กลุ่ม 2 (2.1, 2.2 และ 2.3), 3 (3.1, 3.2 และ 3.3) และ 4 (4.1, 4.2 และ 4.3) ทําการฉีดเชื้อเอ็มจีเสตรน เอฟ, 6/85 และ ของไทยเข้า ไปที่ถุงไข่แดง (yolk sac) 0.1 มล. ตามลําดับ โดยกลุ่มย่อยของแต่ละกลุ่มนั้นมีจํานวน 12 ฟอง และปริมาณเชื้อที่ได้รับลดลง 108, 106 และ 104 ซีเอฟยู/มล. ตามลําดับ สังเกตการตายของคัพภะช่วงแรก (3-6 วันหลังรับเชื้อ) และช่วงท้าย (7 วันหลังรับเชื้อเป็นต้นไป) เมื่อไก่อายุ 7 วัน เจาะเลือดเพื่อตรวจหาแอนติบอดีด้วยวิธี เอสพีเอ และอีไลซา จากนั้นผ่าซากสังเกตคะแนนรอยโรคของถุงลมช่องอกด้วยตาเปล่า และท่อ ลมด้วยจุลพยาธิวิทยา พร้อมทั้งป้ายเชื้อที่บริเวณถุงลมเพื่อตรวจหาสารพันธุกรรมของเชื้อเอ็มจี ด้วยวิธีพีซีอาร์ ผลพบว่า ทุกกลุ่มพบจํานวนไข่ ฟักที่ตายช่วงแรก ระหว่าง 0–4 ฟอง จํานวนไข่ฟักที่ตายช่วงท้าย ระหว่าง 2–7 ฟอง และพบว่า ทุกกลุ่มพบจํานวนลูกไก่ที่ฟักออกมาแล้วรอด ชีวิตช่วงก่อน 6 วันแรก และอยู่รอดครบ 7 วัน ระหว่าง 0–3 ตัว และ 1–9 ตัว ตามลําดับ คะแนนรอยโรคของถุงลมช่องอกด้วยตาเปล่า ของคัพภะที่ตายและลูกไก่ที่รอดชีวิต มีค่าเฉลี่ย ระหว่าง 0–1.33 โดยไม่พบความแตกต่างอย่างมีนัยสําคัญ แต่หากนํากลุ่มทดลองมา เปรียบเทียบกับกลุ่ม 1 พบว่า กลุ่ม 3.2, 3.3, 4.1 และ 4.2 พบความแตกต่างอย่างมีนัยสําคัญ (p<0.05) ส่วนการประเมินรอยโรคของท่อลม ทางจุลพยาธิวิทยา ค่าเฉลี่ย ระหว่าง 0.81–2.56 โดยไม่พบความแตกต่างอย่างมีนัยสําคัญ จํานวนตัวอย่างที่ให้ผลบวกด้วยวิธีทางเอสพีเอและ อีไลซา คือ 0 และ 1 ตัวอย่าง ตามลําดับ และผลบวกด้วยวิธีพีซีอาร์ คือ 0–4 ตัวอย่าง ซึ่งกลุ่ม 1 และ 2.3 นั้นไม่พบสารพันธุกรรมของ เชื้อเอ็มจี จากผลการศึกษาพบว่าเชื้อเอ็มจีสายพันธุ์ที่แยกได้ในประเทศไทยสามารถก่อให้เกิดรอยโรคของถุงลมช่องอกได้และมีความเป็นไปได้ ที่จะนําไข่ไก่ฟักมาศึกษาความรุนแรงของเชื้อเอ็มจีแทนไก่ทดลอง คําสําคัญ: แอนติบอดี ไข่ไก่ฟัก มัยโคพลาสมา กัลลิเซพติกุม พีซีอาร์ คะแนนรอยโรคถุงลมช่องอกและท่อลม ภาควิชาอายุรศาสตร์ คณะสัตวแพทยศาสตร์ จุฬาลงกรณ์มหาวิทยาลัย กรุงเทพฯ 10330 *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction Mycoplasma gallisepticum (MG) infection is known as a chronic respiratory disease (CRD) in avian species (Kleven, 1998; Ley, 2008). Chickens, turkeys, quails, parrots, pheasants, pigeons, and peacocks are the natural hosts of MG infection (Yoder, 1972). The mortality rate is low unless a secondary microorganism infection is present. MG infection causes sneezing, conjunctivitis, airsacculitis, and decreased egg production in affected birds (Ley, 2008). MG organisms of infected birds can be transmitted to the other birds via direct contact, which is, horizontal transmission. In addition, affected breeders can spread MG organisms through their progeny which is called “vertical transmission” (Jordan, 1996). There are several diagnostic methods, clinical signs, histopathology, MG detection and MG serology, all of which are widely used in MG diagnosis (Kleven, 1998). MG detection including MG
culture and isolation and MG polymerase chain reaction (PCR) testing has been used in most MG laboratories (Ley, 2008). Economic losses due to the decrease in egg production in breeders have been estimated at about 21 eggs/bird, or over US$ 100 millions per year for the US poultry industry (Mohammed et al., 1987). Furthermore, their progeny show decreased feed efficiency, high conversion rate, poor carcass quality, and economic losses due to prevention and treatment costs (Ley, 2008). Therefore, MG infection in chickens has been considered as one of the most pathogenic and economic mycoplasma microorganisms of poultry (Ley, 2008). In addition, MG infection has been a great concern in the poultry industry worldwide (Ley, 2008), including Thailand (Pakpinyo and Sasipreeyajan, 2007; Pakpinyo et al., 2008). There are several MG isolates identified in Thai poultry farms including broiler, layers and broiler breeder farms (Pakpinyo and Saipreeyajan, 2007). Some isolates cause the respiratory signs previously
Pakpinyo S. et al./ Thai J Vet Med. 2011. 41(1): 31-36.
described, whereas some isolates do not show any clinical respiratory signs in chickens; which is similar to the study of Ley (2008) describing that isolates and strains of MG have various relative pathogenicities. Hence, further studies of pathogenecity and virulence should be conducted in order to determine precisely each type of the Thai isolates. The pathogenicity of MG isolates and/or strains (certain isolates) can be determined by challenging studies in chickens. Several experimental studies chose to investigate the pathogenicity or virulence of mycoplasma in embryonated chicken eggs (Power and Jordan, 1973; Bradbury and McCarthy, 1983; Levisohn et al., 1985). Levisohn et al. (1985) determined the pathogenicity of virulent strain of MG in embryonated chicken eggs by the numbers of MG organisms. The objectives of this study were to evaluate the pathogenicity of Thai isolated MG inoculated into embryonated chicken eggs, the effects on embryonated chicken eggs and hatched chicks, and the possibility to use embryonated chicken eggs instead of chickens for MG experimental study.
lesions including hemorrhage and size and swabbed (if possible) at the yolk sac membrane or thoracic airsac to determine the presence of the MG DNA by polymerase chain reaction (PCR) technique. After hatching, chickens of each subgroup were separately raised in card boxes and provided with feed and water ad libitum. All birds were observed for respiratory signs including respiratory rales, sneezing, nasal and ocular discharge. At 7 days old, all chickens were bled for MG serology including serum plate agglutination (SPA) and ELISA, then euthanized and necropsied. The necropsied birds were blindly evaluated for gross thoracic airsac lesion score, histopathological tracheal lesion score and simultaneously swabbed at the left side of the thoracic airsac to determine the presence of the MG DNA by PCR technique.
Materials and Methods
Yolk sac inoculation procedure: Yolk sac inoculation followed the procedure reviewed by Senne (1998). Briefly, an eight-day-old healthy embryonated egg was candled, the air cell and embryo were located, the egg shell was sanitized, a needle was inserted through the top and center of the air cell side up, and the pore was closed with candle wax. The inoculated egg was discarded if the embryo died prior to 48 hours.
Fertile chicken eggs: One hundred and forty fertile chicken eggs were provided by commercial broiler breeder flock free of mycoplasma infection. The egg shells were cleaned and sanitized with 70% ethyl alcohol. After cleaning and sanitizing, these fertile eggs were placed in the egg incubators for 8 days. At the eighth day of incubation, all eggs were candled to observe alive embryonated eggs. For the infertile, unhealthy or dead embryonated eggs, the egg yolks were swabbed to detect DNA of MG and M. synoviae (MS) by polymerase chain reaction (PCR). MG inoculums: MG organisms were propagated from cultured broth stored at -80oC at Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University. MG strains including F and 6/85 (derived from vaccine strains), and Thai isolated were cultured, propagated, and diluted to make various amount for inoculation in eggs of different groups and subtypes. Experimental designs: Twelve healthy embryonated eggs were inoculated with 0.1 ml of Frey’s broth medium via yolk sac route, serving as Group 1 (sham inoculated control). One hundred and eight healthy embryonated eggs were equally divided into 3 groups, each group consisting of 3 subgroups. Group 2, 3 and 4 were inoculated with 0.1 ml of various strains of MG organisms including F, 6/85 and Thai isolated, respectively. Each subgroup was defined as 1, 2 and 3 and had different doses of inocula, which were 108, 106 and 104 colony forming unit (CFU)/ml, respectively. Each dilution of MG inoculum was injected into yolk sacs. The inoculated eggs were identified and placed into the 4 setters depending on the strains of MG and sham inoculated control. All eggs were candled once a day and observed as alive or dead embryos. At 18 days old, all live eggs were transferred to the hatchery tray. The dead embryonated eggs were necropsied to observe gross
Classification of dead embryonated eggs: The dead embryonated eggs were observed as an early or late death (modified from Levisohn et al., 1985). The early or late death was the embryo found dead during 3-6 days or 7-11 days post inoculation, respectively.
Airsac lesion score: The airsac lesion score was grossly evaluated by the following criteria (Kleven et al., 1972): 0: No airsac lesion is observed, 1: Lymphofollicular lesions or slight cloudiness of the airsac membrane are found., 2: Airsac membrane is slightly thick and usually presents small accumulations of cheesy exudates., 3: Airsac membrane is obviously thick and meaty in consistency, with large accumulations of cheesy exudates in one airsac., 4: Lesions are observed the same as 3, but 2 or more airsacs are found. Tracheal lesion score: The tracheal lesion score was microscopically evaluated as the following criteria (Yagihashi and Tajima, 1986): 0: No significant changes are observed., 1: Small aggregate of cells (mainly lymphocytes) is found., 2: Moderate thickening of the wall due to the cell infiltration, and edema commonly accompanied with epithelial degeneration and exudation is present., 3: Extensive thickening of the wall due to the cell infiltration with or without exudation is determined.
MG serology SPA procedure: Fresh sera were tested with MG antigen (Nobilis®, Intervet International BV, Holland) following the manufacturer’s instructions. Briefly, thirty µl of serum were mixed with 30 µl of antigen then incubated at room temperature for 1-2 min before the result could be observed. Negative and positive sera were also included in each test. Sera were then stored at -20oC for ELISA determination.
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ELISA: Frozen sera were completely thawed at room temperature (25oC) before testing. All procedures were done at room temperature. Sera were tested with commercial test kits, ProFLOK® (Synbiotics Corporation, USA) following the manufacturers’ directions. Briefly, diluted sera were added into MG antigen-coated plate, incubated, washed then peroxidase-labeled anti-chicken antibody (conjugated antibody) was added. After incubation, the plate was washed, then a substrate was added, and finally, a stop solution was added. The plate was read in an ELISA reader at 405 nanometer manufactured by Labsystems Multiskan MG Type 352, Finland. The optical density of the negative and positive controls and the samples was calculated, then interpreted according to the manufacturers’ recommendation. For the interpretation of ELISA, titer levels 0-148, 149-743, and equal or higher than 744 are negative, suspicious and positive reactors, respectively.
MG DNA detection PCR procedure: The broth sample was individually determined in this study. This method was described by Lauerman (1998). Briefly, the broth was extracted for DNA template by centrifugation at 15,000xg, washed with distilled water, followed by dilute pellet with distilled water, boiled for 10 min, and then placed at -20oC for 10 min, ending with centrifugation and collection of the supernatant at -20oC until use. For PCR mixture in 50 µl volume, KCl 500 mM, TrisHCl (pH 8.3) 100 mM, dNTP (Fermentas) 1 mM, primer F (5’ GAGCTAATCTGTAAAGTTGGTC 3’) and primer R (5’ GCTTCCTTGCGGTTAGCAAC 3’) (Qiagen) 10 pmole each, Taq polymerase (Fermentas) 1.25 U and DNA template 5 µl (250 ng). MG strain S6 (ATCC 15302) was used as positive control. PCR mixtures were amplified in a DNA thermal cycler (PCR Sprint, Thermo Electron Corporation, Milford, MA) at 94oC for 30 sec, 55oC for 30 sec and 72oC for 60 sec for 40 cycles and followed by 72oC for 5 min. The
PCR product was analyzed in 2% agarose gel (Pharmacia Biotech AB, Uppsala, Sweden), stained with ethidium bromide, visualized by UV transilluminator, and photographed. Statistical analysis: The gross airsac and histopathological tracheal lesion scores were determined by using Chi square test at p < 0.05. Significant difference between the sham negative control group and the treatment groups was analyzed by Mann-Whitney U test. All statistical analyses were tested by SPSS for Windows version 17.0.
Results For the infertile, unhealthy or dead embryonated eggs prior to 8 days of incubation, the egg yolks could not detect the MG and MS DNA. This study revealed that the early and late deaths were found ranging from 0-4 and 2-7 embryonated eggs, respectively. The survival chicks prior to 7 days old and at 7 days old were 0-3 and 1-9 birds, respectively, nothing that group 2.3 had the lowest survival at 7 days old. The dose of inoculum of all strains of MG did not apparently affect the survival chicks (Table 1). The average of gross thoracic airsac lesion score and histopathological tracheal lesion score of the dead embryos and survival chicks ranged from 0-1.33 and 0.81-2.56, respectively, without significant difference; however, a significant difference was observed only in the gross airsac lesion score between group 1 and the treatment groups including 3.2, 3.3, 4.1 and 4.2 (Table 2). The numbers of positive samples against SPA test, ELISA and MG PCR were 0, 1 and 0-4, respectively. MG DNA could not be detected in group 1 and 2.3 (Table 3). In addition, MS DNA was not found in the survival chicks at 7 days old of all groups.
Table 1 Numbers of embryonated egg deaths during early and late death period and numbers of dead chicks prior to 7 days old and survival chicks at 7 days old Group 1 (Neg.) 2.1 (F,107) 2.2 (F,105) 2.3 (F,103) 3.1 (6/85,107) 3.2 (6/85,105) 3.3*(6/85,103) 4.1 (Thai,107) 4.2 (Thai,105) 4.3 (Thai,103)
Number of embryonated egg deaths Early death Late death 1 4 1 4 1 1 1 0 1 3
2 4 6 7 4 3 4 2 2 2
Number of chick deaths prior to 7 days old
Number of survival chicks at 7 days old
0 2 0 0 0 0 0 1 3 1
9 2 5 1 7 8 6 9 6 6
*One embryonated eggs was discarded due to death before 48 hours; the total numbers was 11 embryonated eggs.
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Table 2: Blind evaluation of gross thoracic airsac and histopathological tracheal lesion scores (n: numbers of pooled samples of dead embryos and survival chicks). Group
Table 3: Numbers of positive samples against SPA test, ELISA and MG DNA positive samples by PCR Numbers of positive reactors SPA ELISA 1* 0/9 0/9 2.1 0/2 0/2 2.2* 0/5 1/5 2.3* 0/1 0/1 3.1 0/7 0/7 3.2 0/8 0/8 3.3 0/6 0/6 4.1 0/8 0/8 4.2 0/5 0/5 4.3 0/6 0/6 *MG PCR was tested only on survival chicks at 7 days old Group
Discussion This study revealed that all strains and Thai isolated of MG caused the death of embryonated eggs and the lesions of the thoracic airsac including presence of the blood vessels, cloudy, thickening and caseous mass. In addition, for the survival chicks at 7 days old, the gross thoracic airsac and histopathological tracheal lesion scores did not depend on the dose of inoculum. The study found that the F strain caused the highest embryo mortality, followed by 6/85 strain and Thai isolated, respectively. Generally, the F strain is more virulent vaccine compared with ts-11 or 6/85 strains (Whithear, 1996). However, the virulence of MG Thai isolated was similar to that of 6/85 in cases that the data of survival chicks were considered. Three deaths of embryonated eggs of the sham negative control were found without remarkable lesion or bacterial growth suggesting the normal death of embryos. The MG Thai isolated used in this study showed less pathogenicity compared with in vivo pathogenicity (Pakpinyo, 2005). From our previous in vivo study, the MG-inoculated chicks showed severe gross thoracic airsacs and histopathological tracheal lesion scores, and high mortality. Interestingly, this study had a similar result to Levisohn et al. (1985) in that there is no or only a little relation between in ovo and in vivo pathogenicity study. Furthermore, Levisohn et al. (1985) found that the numbers of
Numbers of MG DNA positive samples by PCR 0/9 1/4 4/5 0/1 1/10 1/11 1/9 4/12 2/10 2/7
embryo mortality did not have a correlation with the dose of MG inoculation, which corresponded to our study. The reason of low dose of MG inoculation causing the higher numbers of embryos deaths during the late period was possibly due to the enormous growth of MG microorganisms in the yolk even though non virulent strain leading to embryo deaths (Lin and Kleven, 1984; reviewed by Levisohn et al., 1985). The serological results of the treatment groups found only 1 positive reactor against ELISA out of 48 samples. This was possibly due to the early stage of immune response of hatched chick; if this study was extended, the numbers of positive reactors would surely increased. Interestingly, all 48 samples of the treatment groups were not found positive by the SPA test possibly due to what was previously described. Actually, this procedure should detect the positive reactor from this study because SPA test detects immunoglobulin (Ig) M, which is the first Ig to be formed at approximately 7-10 days after infection (Kleven, 1975; Kleven, 1981). However, almost all of the treatment groups presented the MG DNA, except group 2.3, which had only 1 survival chick left to be performed by PCR. For the positive MG DNA sample, the present study suggested that the gross thoracic airsac lesion scores equal or higher than 2 appeared to be positive by PCR, which was useful for the servicemen or veterinarians to diagnose MG. However, the airsacculitis can be found in other infectious diseases including Mycoplasma synoviae
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infection (Kleven and Ferguson-Noel, 2008), colibacillosis (Barnes et al., 2008), Ornithobacterium rhinotracheale infection (Chin et al., 2008) etc. The present study reveals that the evaluation of the virulence or pathogenicity of Thai isolated MG by using embryonated eggs is possible. There are several advantages of using embryonated eggs including no requirement of experimental chickens and rooms, shorter duration compared with in vivo study, uncomplicated rearing etc. Conversely, several aspects should be concerned including the quality of embryonated eggs, none of the maternal-derived antibody against MG, the adequate capacity to place eggs in the same incubator, the ambiguous classification during egg candle between dead or alive embryonated eggs etc. Therefore, the use of embryonated eggs instead of chickens is possible unless these weak aspects have not been managed.
Acknowledgement This study was supported by the Grants for Veterinary Science Research Fund 2009.
References Bradbury, J.M. and McCarthy, J.D. 1983. Pathogenicity of Mycoplasma iowae for chick embryos. Avian Pathol. 12: 483-496. Barnes, H.J., Nolan, L.K. and Vaillancourt, J.P. 2008. Colibacillosis In: Diseases of Poultry. 12th ed. Y.M. Saif, et al. (eds.) Blackwell Publishing Professional, Ames, IA: 691-737. Chin, R.P., van Empel, P.C.M. and Hafez, H.M. 2008. Ornithobacterium rhinotracheale infection In: Diseases of Poultry. 12th ed. Y.M. Saif, et al. (eds.) Blackwell Publishing Professional, Ames, IA: 765-774. Jordan, F.T.W. 1996. Avian Mycoplasmosis In: Poultry Disease. 4th ed. F.T.W. Jordan and M. Pattison (eds.) W.B. Saunders Company Ltd. London: 81-93. Kleven, S.H., King, D.D. and Anderson, D.P. 1972. Airsacculitis in broilers from Mycoplasma synoviae: effect on air-sac lesions of vaccinating with infectious bronchitis and Newcastle virus. Avian Dis. 16: 915-924. Kleven, S.H. 1975. Antibody response to avian mycoplasmas. Am J Vet Res. 36: 563-565. Kleven, S.H. 1981. Transmissibility of the F strain of Mycoplasma gallisepticum in leghorn chickens. Avian Dis. 25: 1005-1018. Kleven, S.H. 1998. Mycoplasmosis. In: A Laboratory Manual for the Isolation and Identification of Avian Pathogens. 4th ed. D.E. Swayne, J.R. Glisson, M.W. Jackwood, J.E. Pearson, W.M.
Reed (eds). American Association of Avian Pathologists, Kennett Square, PA USA: 74-80. Lauerman, L.H., 1998. Mycoplasma PCR assays. In: Nucleic and Amplification Assays for Diagnosis of Animal Diseases. L.H. Lauerman (ed.). Turlock, CA, American Association of Veterinary Laboratory Diagnosticians: 41-42. Ley, D.H. 2008. Mycoplasma gallisepticum infection In: Diseases of Poultry. 12th ed. Y.M. Saif, et al. (eds.) Blackwell Publishing Professional, Ames, IA: 807-834. Levisohn, S., Glisson, J.R. and Kleven, S.H. 1985. In ovo pathogenicity of Mycoplasma gallisepticum strains in the presence and absence of maternal antibody. Avian Dis. 29: 188-197. Lin, M.Y. and Kleven, S.H. 1984. Evaluation of attenuated strains of Mycoplasma gallisepticum as vaccines in young chickens. Avian Dis. 28: 88-99. Mohammed, H.M., Carpenter, T.E. and Yamamoto, R. 1987. Economic impact of Mycoplasma gallisepticum and M. synoviae in commercial layer flocks. Avian Dis. 31: 477-482. Pakpinyo, S. 2005. The virulence of Mycoplasma gallisepticum infections using Thai isolates in broiler chickens. Thai J Vet Med 35: 21-30. Pakpinyo, S. and Sasipreeyajan, J. 2007. Molecular characterization and determination of antimicrobial resistance of Mycoplasma gallisepticum isolated from chickens. Vet Microbiol. 125: 59-65. Pakpinyo, S., Rawiwet, V., Buranasiri, W. and Jaruspibool, S. The efficacy of Tilmicosin against brolier chickens infected with Mycoplasma gallisepticum isolated in Thailand. Thai J Vet Med. 38: 17-24. Power, J. and Jordan, F.T.W. 1973. The virulence of Mycoplasma gallisepticum for embryonated fowl eggs. Res Vet Sci. 14: 259-261. Senne, D.A. 1998. Virus propagation in embryonating eggs. In: A Laboratory Manual for the Isolation and Identification of Avian Pathogens. 4th ed. D.E. Swayne, J.R. Glisson, M.W. Jackwood, J.E. Pearson and W.M. Reed (eds.) American Association of Avian Pathologists. Kennett Square, PA, USA: 235-240. Whithear, K.G. 1996. Control of avian mycoplasmoses by vaccination. Rev Sci Tech. 15: 1527-1553. Yagihashi, T. and Tajima, M. 1986. Antibody responses in sera and respiratory secretions from chickens infected with Mycoplasma gallisepticum. Avian Dis. 30: 543-550. Yoder, H.W. 1972. Avian Mycoplasmosis In: Diseases of Poultry. 6th ed. M.S. Hafstad et al. (eds.) The Iowa State University Press, Ames, IA: 282-301.
Clinical Study on the Treatment of Piroline against Bovine Mastitis Jian-Ping Liang1,2 Bao-Cheng Hao1* Xue-Hong Wang1 Zhi-Ting Guo1 Wen-Zhu Guo1 Ruo-Feng Shang1 Lei Tao1 Yu Liu1 Zhao-Zhou Li1 Lan-Ying Hua1 Shu-Yang Wang3
Abstract The study aims to investigate the efficacy of piroline and antibiotics in the treatment of bovine mastitis caused by Streptococcus uberis (S. uberis) and Escherichia coli (E. coli) during dry-milk period. 1880 cows in dry-milk period were divided into 4 groups and treated with penicillin G, ammonia benzyl penicillin, ceftiofur and piroline, respectively. The efficacy of each medicine in treating mastitis caused by E. coli intramammary infection (E. coli IMI) was followed: 31.2% for penicillin G, 36.9% for ammonia benzyl penicillin, 61.3% for ceftiofur, and 64.4% for Piroline. For those caused by S. uberis intramammary infection (S. uberis IMI), the efficacy of ceftiofur was 90% and piroline was 94.4%. The results indicated that piroline was more effective than the other three in treating the disease. The following analysis on milk samples demonstrated that there was no piroline residue in those treated cows’ milk. Based on these data, it can be predicted that piroline will have a bright future in treating cow intramammary mastitis. Keywords: bovine mastitis, efficacy, piroline 1Key
Laboratory of New Animal Drug Project of Gansu Province/Key Laboratory of New Animal Drug Project of CAAS, Lanzhou Institute of Animal Science and Veterinary Pharmaceutics, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China 2Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730050, China. 3Lanzhou University, Lanzhou 730000, China *Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 39-43.
Liang J. et al. / Thai J Vet Med. 2011. 41(1): 39-43.
บทคัดย่อ การศึกษาทางคลินิกของ Piroline ในการรักษาโรคเต้านมอักเสบในโคนม Jian-Ping Liang1,2 Bao-Choog Hao1* Xue-Hong Wang1 Zhi-Ting Guo1 Wen-Zhu Guo1 Ruo-Fong Shang1 Lei Tao1 Yu Liu1 Zhao-Zhao Li1 Lan-Ying Hua1 Shu-Yang Wang3 จุดประสงค์ในการศึกษาครั้งนี้ เพื่อศึกษาประสิทธิภาพของ Piroline และยาปฏิชีวนะในการรักษาโรคเต้านมอักเสบในโคนมที่เกิด จากการติดเชื้อ Streptococcus uberis (S. uberis) และ Escherichia coli (E. coli) ในช่วงหยุดให้นม โคนมจํานวน 1880 ตัวถูกแบ่งเป็น 4 กลุ่มทดลอง และทําการรักษาด้วยยา Penicillin G, ammonium benzyl penicillin, ceftiofur และ Piroline ประสิทธิภาพของยาทั้ง 4 ชนิด ในการรักษาโรคเต้านมอักเสบที่ติดเชื้อ E. coli (E. coli IM) มีค่าร้อยละ 31.2 ของ Penicillin G, ร้อยละ 36.9 ของ ammonium benzyl penicillin, ร้อยละ 61.3 ของ ceftiofur และร้อยละ 64.4 ของ Piroline ประสิทธิภาพของยาทั้ง 4 ชนิด ในการรักษาโรคเต้านม อักเสบที่ติดเชื้อ S. uberlis (S. uberlis IM) มีค่าร้อยละ 90 ของ ceftiofur และร้อยละ 94.4 ของ Piroline จากผลการทดลองพบว่า Piroline มีประสิทธิภาพที่ดีกว่ายาทั้งสามชนิดในการรักษา ผลการตรวจวิเคราะห์น้ํานม ไม่พบสารตกค้างของ Piroline ในน้ํานมของโคที่ รักษา ซึ่งแสดงถึงผลของ Piroline ที่ดีในการใช้เป็นยารักษาโรคเต้านมอักเสบในโคนม คําสําคัญ: โรคเต้านมอักเสบในโคนม ประสิทธิภาพ Piroline 1 Key Laboratory of New Animal Drug Project of Gansu Province/Key Laboratory of New Animal Drug Project of CAAS, Lanzhou Institute of Animal Science and Veterinary Pharmaceutics, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China 2 Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730050, China. 3 Lanzhou University, Lanzhou 730000, China *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction Bovine mastitis is not only a major disease affecting the dairy industry, but also one of the major influencing factors in milk production (Yuan et al., 1992). It causes great economic losses and decreases animal health. Although much progress has been made in control of cow mastitis, producers still cannot prevent and cure it effectively. As reported in a literature (Yuan et al, 1992), 3650 strains of 24 species of bacteria and fungi were isolated and identified from 3006 milk samples, in which 2060 strains of 12 species were closely related to cow mastitis. The isolation rate of pathogenic bacteria was 62.5%. Bacteria related to mastitis were mainly S. uberis (38.11%), E. coli (7.14%), etc. Buddle and Cooper reported (Buddle and Cooper, 1980) that about 32% of found cure spontaneously over the dry period. Dry cow treatment (DCT) is an important step of a mastitis control program, the advantages of DCT include reducing incidence of intramammary infections (IMI) at parturition and increasing cure rate of IMI (Buddle and Cooper, 1980). The aims of DCT are to cure existing intramammary infection (IMI), and to prevent new infection during the dry period.
A previous study that bacteriological cure rates for various intramammary dry cow treatments ranged from 25% to 75%. Systemic DCT was previously studied in an attempt to improve the cure rates. The systemic administration of antibiotics was evaluated in different studies. Owing to antibacterial drug resistance, only a few of antimicrobial agents demonstrated an improved cure rate over conventional intramammary DCT. Piroline is a mainly active component isolated from the Rubia Cordifolia, which has been used in China to treat bovine mastitis for a long time (Liang et al, 1993; Liang et al, 2000). The pharmacological and antibacterial properties suggested that there was much less or no drug resistance to piroline, and thus it might be valuable for the treatment of bovine mastitis. The purpose of the present study was to compare the efficacy of piroline with that of other antibiotics commonly used in the systemic DCT. The results would be helpful for better prevention and cure of bovine mastitis.
Materials and Methods Herds: The DCT field trials were conducted in three factory-supply dairy herds under seasonal-calving
Liang J. et al./ Thai J Vet Med. 2011. 41(1): 37-41. conditions in Lanzhou, Gansu of China. Owners agreed to the following conditions: 1) provide calculated calving dates; 2) schedule dates for drying off, sampling, and treatment; and 3) permit some cows to serve as untreated controls. General information on several herds is presented in table 1. Udder selection and milk sample collection: Udders and milk samples were collected through recommended procedures. Composite milk samples were collected from 1880 lactating cows from three herds within 4 weeks prior to drying off. Duplicate quarter milk samples were collected at drying off. And within 21 days, single sample was collected from all quarters of cows in treatment 1 and 3 at the prepartum period prior to infection of the lactating cow product (LCP). Drugs: Penicillin G (batch number, h0605071) and Ammonium benzyl penicillin (batch number h06050714) was purchased from North China Pharmaceutical Group Corporation, Shijiazhuang city, China. Ceftiofur (batch number y06030914) was from Heibei Yuan Zheng Pharmaceutical Co., Ltd. Shijiazhuang, China. And Piroline was made by Lanzhou Institute of Animal & Veterinary Pharmaceutics Sciences of Chinese Academy of Agricultural Sciences, Lanzhou city, China. Assignment of cows to treatment groups: Cows with E. coli IMI, based on cultural results of composite milk samples, were assigned randomly to the four treatment groups, a minimum of 80 E. coli IMI was included in each of the four treatment groups. More than 90 SU IMI were included initially in treatment 1 and 3 in anticipation of missed infusions due to early calving or incorrect calculated calving dates. Cow missed scheduled prepartum treatment were included in treatments 2 and 4, respectively. All four treatment groups were represented within each herd in table 2. Treatment regimens: Each group of treatments was listed in table 2. Treatment 1 received an infusion at drying off with penicillin G 2,000,000 IU/50 ml (water), treatment 2 with ammonium benzyl penicillin 0.5 g/50 ml (water), treatment 3 with ceftiofur 0.5 g/50 ml (water), and treatment 4 with piroline 0.4 g/50 ml(water) (Brander, 1969). Table 1 Information on cooperation dairy farms Herd
Table 2 Experimental design of dry-cow therapy field trial Time of treatment Drying off Prepartum 1 + + 2 + + 3 + + 4 + + +: Intramammary infections Treatment
41 Microbiological procedures: Method recommended by the National Mastitis Council, U.S.A., was followed. Presumptive identification of the following microorganisms was made; Escherichia coli (E. coli); Staphylococcus epidermidis (SE); Streptococcus galactiae (SAg); Streptococcus uberis (S. uberis); other streptococci (OS); Corynebacterium bovis (CB) and Coliforms (CO). Definition of terms: Infection: the number of somatic cells in the milk samples exceeds regular range and determination of pathogenic bacteria in the milk shows positive. Cure: clinical symptoms ease off or disappear, the number of somatic cells in the milk returns to regular range, and determination of pathogenic bacteria in the milk shows negative. Fail: clinical symptoms do not ease off, the number of somatic cells in the milk does not return to regular range, and determination of pathogenic bacteria in the milk shows positive. Statistical analysis: An analysis of variance was conducted only on the E. coli and S. uberis data. The number of IMI with other pathogens was insufficient to conduct valid analysis. Developed (%) = (developed quarters/treated quarters) x 100%, and Cured% = (cured quarters/treated quarters ) x 100%.
Results The efficacy was 61.3% against S. uberis IMI in treatment 3 and 64.4% in treatment 4 (Table 3), and the difference was not significant. The efficacy of treatment 4 was significantly greater (p<0.01) than the 36.9% in treatment 2, and treatment 4 was also significantly different from treatment 1 (31.2%, (p<0.01). Treatment 4 and 3 were significantly different from treatment 1 and 2. Approximately 10.5% of quarters developed from new IMI with E. coli or S. uberis during the dry period (Table 4). These resulting pathogens were accounted for 93% of new IMI. Other new IMI were caused by SAg and OS, about 3% for each one. Though differences were observed between the treatment groups, the incidence of the new IMI was similar to E. coli and S. uberis. In treatment 1 and 2, the incidence of IMI with S. uberis almost doubled than that in treatments 3 and 4 (Liang et al., 1993; Liang et al., 2000). Efficacy of prepartum treatment with the ceftiofur and piroline against new SAg IMI was low, but it was 61.3% and 64.4% against the new IMI with S. uberis respectively (Table 3). The rate of new dry period IMI with SU ranged from 3.6% to 4.9% of quarters for treatment 1, 2 and 3. 10.5% of quarters become infected with SU in treatment 4 (Table 4), prior prepartum samples were analyzed from cows in treatment 3 and 4 , so efficacy of ceftiofur and piroline were excellent, 90% and 94.4%, respectively for treatment 3 and 4. No prepartum samples were analyzed from cows in treatment 1 and 2 and spontaneous recoveries were determined for the entire dry period using samples collected postpartum.
Liang J. et al. / Thai J Vet Med. 2011. 41(1): 39-43.
Table 3 Efficacy of dry-cow therapy against S. uberis
1 2 3 4 Totals
91 102 83 98 374
243 288 211 270 1012
Quarter Number Intramammary infections Drying off Postpartum 99 61 102 64 89 66 91 70 381 261
Efficacy (%) 31.2 36.9 61.3 64.4
Table 4 New dry-period intramammary infections Number.
S. uberis Quarter Quarter Cows Quarters Developeda Curedb Developeda Number Number 1 98 270 21 7.8 0 10 3.9 2 102 288 10 3.6 0 10 3.6 3 91 243 8 3.4 45 12 4.9 4 83 211 15 7 60 22 10.5 Totals 374 1012 54 5.3 54 5.3 aDeveloped after prepartum treatment, bExpressed as percentage cure of IMI that developed prepartum sampling. Group
Discussion Treatment 4, treated with piroline, did not significantly reduce the number of E. coli infections postpartum when compared with treatment 3. On the contrary, ceftiofur showed good effect in reducing the number of E. coli IMI significantly when compared with treatment 1 and 2. Treatment 4, treated with piroline, did not significantly reduce the level of E. coli IMI only with ammonium benzyl penicillin for prepartum treatment as compared with penicillin. The rate of spontaneous recovery for E. coli during dry period was consistent with the figures previously reported. Efficacy of piroline was 91.3% when compared with other reports on piroline against E. coli. The range in efficacy among herds was from 83% to 95%, which corresponded with earlier studies (Liang et al., 1993). The rate of new E. coli dry period IMI could be reduced 50% by DCT and supported earlier work. The incidence of new IMI with S. uberis was similar to that observed with E. coli and was reduced about 50% by DCT (Brander, 1969; Brown et al, 1969; Christie et al, 1974；Buddle and Cooper, 1980). However, the incidence of new S. uberis IMI was similar to that in treatment 3 and 4. Further studies are required to explain these results since no prepartum samples were collected and detected from cows in treatment 1 and 2. Additionally, the rate of spontaneous recovery was high for new S. uberis IMI during the late dry or early postpartum period. Philpot (Philpot, 1969; Philpot, 1979) reported that spontaneous recovery over the dry period was 70% for Streptococci, compared with 27% for E. coli. Prepartum therapy with piroline was effective in eliminating over 90% of new S. uberis IMI, but it was not effective in eliminating new S. uberis IMI (Liang, 2000). The average efficacy against new E. coli IMI was less than 60%, but the number of new E. coli was largely reduced. A wide variation in the
Curedb 0 0 90 94.4
efficacy was observed when compared with the earlier reports, but these data may not be conclusive. The results validated many earlier reports on piroline reducing the incidence of new dry period IMI with E. coli and S. uberis. Prepartum therapy with piroline appeared to be of marginal benefit and probably would be of practical value in dairy herds experiencing significant clinical mastitis cows. Philpot reported a 68% reduction of clinical cases during the first week of lactation when cows received penicillin G at parturition and the end of lactation. Results from this field trial provided supportive evidence of the effectiveness of piroline, eliminating many new IMI by educing levels of infected quarters before and after parturition, especially for the IMI infected with S. uberis.
Acknowledgement The present study was supported by The Institute of Traditional Chinese Veterinary, Chinese Academy of Agricultural Science and National Nature Science Foundation of China (grant No. 10275084). The authors are very grateful to professor Li Zhang and Yanbin Si for assistance in experimental design and review of manuscript.
References Wilson, C.D. 1964. The use of antibiotics in the control of mastitis. Proceedings of the Royal Society of Medicine 12(57): 1088-1090. Brown, R.W, Morse, G.E and Newbould, F.H.S, 1969. Microbiological Procedures for the Diagnosis of Bovine Mastitis. National Mastitis Council, Washington DC. Buddddie, G.E. and Cooper. 1980. Dry-cow therapy for Staphylococcus aureus mastitis. New Zealand Vet J. 28: 51-53. Christie, G.J, Keefe, T.J and Strom, P.W. 1974. Cloxacillin and the dry cow. Vet Med Small
Liang J. et al./ Thai J Vet Med. 2011. 41(1): 37-41. Anim Clin. 69: 1403-1408. Dodd, F.H. and Griff, T.K. 1975. The role of antibiotic treatment at drying off in the control of mastitis. In Proceedings of the Int Dairy Fed Seminar on Mastitis Control. Reading, UK: 282-302. Heald, C.W., Jones, G.M., Nickerson, S. and Bibb, T. L. 1977. Mastitis control by penicillin and novobiocin at drying off. Can Vet J. 18: 171-179. Kingwill, R.G., Neave, F.K., Dodd, F.H., Griffin, T. K., Westgarth, D. R. and Wilson, C. D. 1970. The effect of a mastitis control system on levels of subclinical and clinical mastitis in two years. Vet Rec. 87: 94-99. Liang, J.P., Wei, Z.Q., Zhang, L., and Yu, J., 2000. Use of Piroline for treatment of bovine mastitis. Indian Vet J. 77: 553. Liang, J.P., Zhang, J.Y., Zhao, R.C., Xu, Z.Z., Li, J.S. and Yu, J. 1993. Preparation and application of piroline in veterinary clinic. China J Vet Sci Tech. 12(23): 14-16 .
43 Natzke, R.P. 1971. Therapy: One component in a mastitis control system. J Dairy Sci. 54: 18951901. Philpot, W.N. 1979. Control of mastitis by hygiene and therapy. J Dairy Sci. 62: 168-176. Philpot, W.N. 1969. Role of therapy in mastitis control. J Dairy Sci. 52: 708-713. Smith, A., Rautenbach, H.F.P., Dodd, F.H. and Bander, G.C. 1975. The effect of udder infection of varying the levels and persistency of antibiotic in the dry period. In Proceedings I Int Dairy Fed Seminar on Mastitis Control, Reading. UK: 345-348. Swenson, G.H. 1979. Posology and field efficacy study with novobiocin for intramammary infusion in nonlactating dairy cows. Canadienne De Médecine Comparée. 43: 440-447. Yuan, Y.L, Zhang, L.H. and Liu, C.C. 1992. A survey of the pathogens of bovine mastitis in China. Scientia Agriculturea Sinica 25(4): 70-76.
Chronic Toxicity Study of Garcinia mangostana Linn. pericarp Extract Songpol Chivapat1* Pranee Chavalittumrong1 Prapai Wongsinkongman1 Chada Phisalpong2 Anudep Rungsipipat 3
Abstract Ethanolic extracts from fruit pericarp of mangosteen (Garcinia mangostana L.) possess many biological and pharmacological activities. However, chronic toxicity study of ethanolic extract has never been investigated. The objective of this study was to evaluate the safety of 95% ethanolic extract from mangosteen pericarp in animal model. The oral administration of the extract was performed in 180 Wistar rats randomly allocated to six groups, each of 15/sex. Group 1 was control group receiving distilled water. Group 2 to 6 were treatment groups receiving the extract at the doses of 10, 100, 500, 1000 and 1000 mg/kg/day for six months respectively. The last group was assigned to be the satellite group for the study of reversibility of the extract effects after two-week of extract withdrawal. The results revealed that the highest dose extract produced significantly lower body weights in both male and female rats, compared to their corresponding control groups. The extract at any tested doses did not affect the animals’ behavior, health status and nor did produce any abnormality of clinical manifestations and hematological values. Clinical chemistry results showed that the male rats treated with 500 mg/kg/day extract onward had significantly higher ALT than their control group. Both male and female receiving the highest dose extract had significantly higher AST, whereas their glucose levels were significantly lower when compared to their corresponding control groups. The male rats of the highest dose and satellite groups had significantly higher BUN values than their control group. The female rats receiving the extract at the dose of 500 mg/kg/day onward had significantly higher BUN and creatinine values than their control group. Histopathological results of visceral organs revealed no significant lesion related to the extract; except the satellite group of both sexes, which had significantly higher lesion of centrilobular hydropic degeneration in their livers than the corresponding control groups. Such alteration may be caused by the highest dose mangosteen pericarp extract. In conclusion, the high dose mangosteen pericarp extract affected liver and kidney. Safety of chemical constituents in the extract should be further investigated before the usage for health promotion. Keywords: chronic toxicity, mangosteen pericarp extract, rat 1Medicinal
Plant Research Institute, Department of Medical Sciences, Muaeng District, Nonthaburi Province, Thailand 11000 of Research and Development, Government Pharmaceutical Organization, Bangkok, Thailand 10400 3Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn university, Bangkok, Thailand 10330 *Corresponding author E-mail: [email protected]l.go.th 2Institute
Thai J Vet Med. 2011. 41(1): 45-53.
Chivapat S. et al. / Thai J Vet Med. 2011. 41(1): 45-53.
บทคัดย่อ การศึกษาพิษเรื้อรังของสารสกัดเปลือกมังคุด ทรงพล ชีวะพัฒน์ 1* ปราณี ชวลิตธํารง 1 ประไพ วงศ์สินคงมั่น 1 ชฏา พิศาลพงศ์ 2 อนุเทพ รังสีพิพัฒน์ 3 สารสกัดเปลือกมังคุดมีฤทธิ์ทางชีวภาพและเภสัชวิทยาที่น่าสนใจหลายประการ แต่ยังไม่มีรายงานการศึกษาพิษเรื้อรัง การศึกษา ครั้งนี้มีวัตถุประสงค์ เพื่อให้ทราบถึงความปลอดภัยของสารสกัดเปลือกมังคุดด้วยเอทานอล โดยวิธีป้อนสารสกัดทางปากแก่หนูแรทพันธุ์ วิสตาร์ จํานวน 180 ตัว แบ่งออกเป็น 6 กลุ่มๆละ 30 ตัว (เพศละ 15 ตัว) ดังนี้ กลุ่มที่ 1 กลุ่มควบคุมได้รับน้ํากลั่น กลุ่มที่ 2 ถึง 6 เป็นกลุ่ม ทดลอง ที่ได้รับสารสกัดเปลือกมังคุดขนาด 10, 100, 500,1000 และ 1000 มก./กก./วัน เป็นเวลา 6 เดือนตามลําดับ โดยกลุ่มสุดท้ายเป็น กลุ่มศึกษาผลย้อนกลับ (satellite group) ภายหลังหยุดให้สารสกัดเป็นเวลา 2 สัปดาห์ ผลการศึกษาพบว่า สารสกัดเปลือกมังคุดขนาด 1000 มก./กก./วัน ทําให้หนูเพศผู้และเพศเมีย มีน้ําหนักตัวต่ํากว่ากลุ่มควบคุมอย่างมีนัยสําคัญ (p<0.05) สารสกัดทุกขนาดไม่มีผลต่อ พฤติกรรม สุขภาพ รวมทั้งไม่ทําให้หนูมีอาการแสดงออกและค่าทางโลหิตวิทยาผิดปกติแต่อย่างใด การตรวจค่าทางเคมีคลินิก พบว่า หนูเพศ ผู้ที่ได้รับสารสกัดตั้งแต่ 500 มก./กก./วันขึ้นไป มีค่าเอนไซม์ ALT สูงกว่ากลุ่มควบคุมอย่างมีนัยสําคัญ (p<0.05) หนูเพศผู้และเพศเมียที่ได้รบั สารสกัดขนาด 1000 มก./กก./วัน มีเอนไซม์ AST สูงขึ้นอย่างมีนัยสําคัญ (p<0.05) แต่มีระดับกลูโคสลดลงอย่างมีนัยสําคัญ (p<0.05) เมื่อ เปรียบเทียบกับกลุ่มควบคุม หนูเพศผู้ที่ได้รับสารสกัดขนาด 1000 มก./กก./วัน และกลุ่ม satellite มีค่า BUN สูงกว่ากลุ่มควบคุมอย่างมี นัยสําคัญ (p<0.05) ส่วนหนูเพศเมียกลุ่มที่ได้รับสารสกัดตั้งแต่ 500 มก./กก./วัน ขึ้นไปมีค่า BUN และ creatinine สูงกว่ากลุ่มควบคุมอย่าง มีนัยสําคัญ (p<0.05) ผลทางจุลพยาธิวิทยาของอวัยวะภายใน ไม่พบรอยโรคใดๆ ที่เกิดจากสารสกัดเปลือกมังคุด ยกเว้นหนูกลุ่ม satellite พบรอยโรคการเสื่อมแบบมีน้ําในเซลล์ตับสูงกว่ากลุ่มควบคุมอย่างมีนัยสําคัญ (p<0.05) ซึ่งอาจเกิดจากสารสกัดเปลือกมังคุดขนาดสูงสุด การศึกษาครั้งนี้สรุปได้ว่า สารสกัดเปลือกมังคุดขนาดสูงมีผลต่อตับและไต หากนําไปใช้เสริมสุขภาพควรมีการศึกษาด้านความปลอดภัยของ องค์ประกอบทางเคมีต่างๆเพิ่มเติมต่อไป คําสําคัญ: พิษเรื้อรัง สารสกัดเปลือกมังคุด หนูแรท 1 สถาบันวิจัยสมุนไพร กรมวิทยาศาสตร์การแพทย์ จ. นนทบุรี 11000 2 สถาบันวิจัยและพัฒนา องค์การเภสัชกรรม กรุงเทพฯ 10400 3 ภาควิชาพยาธิวทิ ยา คณะสัตวแพทยศาสตร์ จุฬาลงกรณ์มหาวิทยาลัย ปทุมวัน กรุงเทพฯ 10330 *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction Fruit pericarp of mangosteen (Garcinia mangostana L.) has been traditionally used for centuries in Southeast Asians as a medicinal agent for the treatment of skin infections, wounds, amoebic dysentery and also inflammation, diarrhea, cholera and dysentery in Ayurvedic medicine. (PedrazaChaverri et al., 2008). Several phytochemical studies have shown that there are many xanthones compounds in the pericarp of mangosteen fruit. For example, α- mangostin, γ-mangostin, 8-deoxygartnin, garcinone E, mangostanol (Chairungsrilerd et al., 1996), tovophillin A and B (Huang et al., 2001), mangostenin (Suksamrarn et al., 2003), mangotenones C, D and E (Suksamrarn et al., 2006). activities
Many biological and of the compounds
pharmacological extracted from
mangosteen pericarp including its crude extracts have been extensively investigated. For instance, αmangostin, the most abundant compound in the pericarp extract, exert antiproliferative activity against human leukemia cells (Matsumoto et al., 2003) and also possess antimalarial properties (Mahabusarakam et al., 2006). Moreover, this compound was shown to possess potent chemopreventive effects in rat colon carcinogenesis (Nabandith et al., 2004). Garcinone E has potent cytotoxic effect on lung, gastric, lung cancer human cell lines (Ho et al., 2002). Suksamrarn et al. (2003) has shown that α-and β-mangostin and garcinone B exhibit strong antituberculosis activity. An ethanolic extract of the mangosteen pericarp was demonstrated to possess antibacterial (Voravuthikunchai and Kitipat, 2005), antioxidant and neuroprotective (Weecharangsan et al., 2005), antiallergy (Nakatani et al., 2002) and antiinflammatory activities in experimental animals (Reanmongkol and Wattanapiromkul, 2008). In
Chivapat S. et al./ Thai J Vet Med. 2011. 41(1): 45-53. addition, it has been reported to exert the remarkable activity against SKBR3 human breast adenoma cell line (Moongkarndi et al., 2004). Even though mangosteen pericarp has been shown to possess various health benefits, the longterm toxic effect of its extract has never been reported. In this study, we investigated chronic toxicity of the mangosteen pericarp extract in experimental animal to gain additional safety information. The results will be beneficial for supporting the development and the consumption of health products from mangosteen pericarp.
Materials and Methods Plant material and preparation of mangosteen pericarp extract (MPE): The fruits of G. mangostana were purchased from Chantaburi Province, Thailand. The voucher specimen (WGM0615) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Thailand. The dried pericarp of mangosteen fruit was coarsely pulverized into powder. The powder was macerated twice with 95% ethanol for 48 and 24 hours respectively. The extract solution from each marceration was filtered and concentrated by evaporation under reduced pressure. Both concentrated extracts were pooled together and heated at 50ºC to remove the solvent. The upper part of the concentrated extract was further evaporated with rotary evaporator at 55ºC under reduced pressure, and dried with vacuum oven at 50ºC for 12 hours. The lower part of the concentrated extract was centrifuged with high-speed centrifuge at 9,000 rpm for ten min, then the supernatant solution was evaporated at 55ºC under reduced pressure using rotary evaporator and then was subjected to vacuum oven at 50ºC for 12 hours. Both concentrated extracts were mixed together at 55ºC until dried. The yield of dried MPE from the dried mangosteen pericarp was about 10% (w/w). Alpha-mangostin, a major biological active compound in MPE, was found to be 24.42% according to HPLC analysis. In addition, total tannins content in MPE was assayed by the applied determination of tannins method described in Thai Herbal Pharmacopoeia Vol II (Department of Medical Sciences, 2000) and it was found to be 13.8% (w/w). The extract was kept in well-closed container, protected from light at -20ºC for further toxicological investigation. Animals: One hundred and eighty Wistar rats (90 male and 90 female rats weighing approximately 180200 and 170-190 g, respectively) were purchased from The National Laboratory Animal Center, Mahidol University. Animals were housed in a hygienic conventional animal room of the laboratory animal center, Department of Medical Sciences where the environment of the room was maintained at 25±1oC with 60% humidity and 12 hour-light-dark cycle. They were raised with commercial pellet diet and clean water ad lib. Prior to the chronic toxicity study, the rats were acclimatized with the environment for two weeks. This study was approved by the Institutional Animal Care and Use Committee, Department of Medical Sciences (Approval No. 49-011).
47 Chronic toxicity test: Wistar rats were randomly allocated to six groups of fifteen animals of each sex. Group1 was control group receiving distilled water at the volume of 10 ml/kg. Group 2 to 6 were experimental groups orally administered with MPE at the doses of 10, 100, 500, 1000 and 1000 mg/kg/day for six months respectively. The last group (satellite group) was further raised without treatment for 14 days, in order to assess reversibility of adverse effects which may be produced by the highest dose extract. During the experimental period, body weight and food intake were recorded weekly and the animals were observed for general appearance, behavior and signs of abnormalities. At the end of the six-month treatment period, the animals were fasted overnight, anesthesized with diethyl ether inhalation. Blood samples were collected from posterior vena cava for determining hematological and serum clinical chemistry values. Hematological analysis was performed using automatic hematological analyzer Cell Dyn® 3500 (Abbot Laboratories Ltd, USA). Parameters examined were red blood cells (RBC), hematocrit (Hct), hemoglobin, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), white blood cells (WBC), neutrophils, eosinophils, lymphocytes, monocytes, basophils and platelets. Clinical chemistry values were measured by using automatic chemistry analyzer Hitachi® 912 (Hitachi Ltd, Japan) and parameters assayed were alkaline phosphatase (ALP), alanine transminase (ALT), aspartate transminase (AST), total protein, albumin, bilirubin, blood urea nitrogen (BUN), creatinine, glucose, uric acid, triglyceride, cholesterol, sodium, potassium and chloride ions. A complete necropsy was performed to determine gross pathological alterations of various visceral organs. Brain, heart, lung, liver, kidney, stomach, spleen, testis, uterus urinary bladder and adrenal glands were weighed by using Mettler Toledo® PB 153 balance (Metler Toledo Int Inc, Switzerland). The organs’ weights were calculated into relative organ weight (g/1000 g body weight). The visceral organs were fixed in 10% buffered formalin, and subjected to conventional histological process. Histopathological examination was performed on the above mentioned organs including the trachea, lymph nodes, esophagus, pancreas, intestine, thyroid gland, lacrimal and salivary gland, prostate gland, seminal vesicle, ovary, uterus, and mammary glands Statistical analysis: The data were statistically evaluated by one way ANOVA. Comparison between treatment and control group were made by Bonferroni test. For histopathological results, Fisher’s exact was applied. Differences between groups were considered significant at p<0.05.
Results Effect of MPE on body weight, food consumption and health status: Male rats receiving MPE at the doses of 1000 and 500 mg/kg/day had significantly lower average body weight than their control group since
Chivapat S. et al. / Thai J Vet Med. 2011. 41(1): 45-53.
the 7th and 17th week till the end of the study respectively. Similar body weight change was observed in the female rats treated with the highest dose MPE at the 12th week onward (Fig. 1). Measurement of the weekly food intake in the male and female over the whole experimental period showed no significant difference between all the treatment groups and their corresponding control
groups in almost every week. Only the male and female rats receiving the highest dose had significantly lower food intake than their corresponding control groups at week 8, 9 and 14 in the former group and at week 2 in the latter group (Fig. 2). All of the MPE-treated groups revealed healthy and no sign of abnormality, as compared to their control groups.
700 Male 600
Body weight (g)
M-10 mg/kg/day M-100 mg/kg/day M-500 mg/kg/day
F-water F-10 mg/kg/day
F-100 mg/kg/day F-500 mg/kg/day
Group receiving 1000 mg/kg/day was significantly different from control group (p<0.05)
Group receiving 500 mg/kg/day was significantly different from control group (p<0.05)
Figure 1 Growth curves of male and female rats receiving MPE for 6 months. 30
Food consumption (g/rat/day)
M-1000 mg/kg/day a
F-10 mg/kg/day F-100 mg/kg/day F-500 mg/kg/day
Significantly different from control group (p<0.05)
Figure 2 Food consumption of male and female rats receiving MPE for 6 months. Table 1 Relative organ weight (g/1000 g of body weight) and body weight (g) of male rats receiving MPE for 6 months Organs Brain Heart Lung Liver Stomach Spleen Right kidney Left kidney Right testis Left testis Right adrenal Left adrenal Bladder Initial body weight Final body weight
Chivapat S. et al./ Thai J Vet Med. 2011. 41(1): 45-53. Effect of ME on relative organ weight: In the male, relative weight of the brain, lung, stomach, both kidneys and right testis in the group receiving 500 mg/kg/day of MPE were significantly higher than those in the control group. Almost all organs in the highest dose group had significantly higher relative weight than those of their control groups except bladder. Similar changes were observed in the satellite group except for adrenal glands and bladder
49 (Table 1). In the female rat, relative stomach weight in the groups treated with 500 mg/kg/day MPE onward was significantly higher than that in the control group. The relative weight of the liver and both kidneys in the highest dose and satellite groups were significantly higher than those in the control group. In addition, the highest dose group had significantly higher relative brain weight than the control group (Table 2).
Table 2 Relative organ weight (g/1000g body weight) and body weight (g) of female rats receiving MPE for 6 months Organs Brain Heart Lung Liver Stomach Spleen Right kidney Left kidney Right adrenal Left adrenal Bladder Uterus Right ovary Left ovary Initial body weight Final body weight
The values are expressed as mean±SD, 1000-S: the satellite group *significantly different from control group (p<0.05)
Effects of ME on hematological values: As depicted in Table 3 and 4, eosinophils in both male and female rats receiving MPE at the doses of 500 and 1000 mg/kg/day were significantly lower than those in their corresponding control groups. Neutrophils in the male rats of highest dose and satellite groups were significantly higher than those in the control group. In addition, WBC in the female rats of the satellite group was significantly higher than that in the control group. Effects of ME on clinical chemistry values: In the male rats, the groups receiving MPE at the doses of
500 and 1000 mg/kg/day had significantly higher ALT than the control group. AST and BUN in the highest dose and satellite group were significantly higher than those in the control group. Cholesterol of the group receiving MPE at the doses of 500 and 1000 mg/kg/day and of the satellite group showed significantly higher level than that of the control group. Total protein, uric acid and glucose in the highest dose group were significantly lower than those in the control group (Table 5). In the female, the highest dose group had significantly higher AST and total bilirubin than the control, whereas the glucose level was significantly lower than that in the control
Table 5 Biochemical values of male rats receiving MPE for 6 months Parameters ALP (U/L ) ALT (U/L) AST (U/L) Total protein (g/dl) Albumin (g/dl) Total bilirubin(mg/dl) BUN (mg/dl) Creatinine (mg/dl) Glucose (mg/dl) Uric acid (mg/dl) Triglyceride(mg/dl) Cholesterol (mg/dl) Sodium Potassium Chloride
The values are expressed as mean±SD, 1000-S: the satellite group * significantly different from control group (p<0.05)
Table 6 Biochemical values of female rats receiving MPE for 6 months
Parameters ALP (U/L ) ALT (U/L) AST (U/L) Total protein (g/dl) Albumin (g/dl) Total Bilirubin(mg/dl) BUN (mg/dl) Creatinine (mg/dl) Glucose (mg/dl) Uric acid (mg/dl) Triglyceride (mg/dl) Cholesterol (mg/dl) Sodium Potassium Chloride
The values are expressed as mean±SD, 1000-S: the satellite group * significantly different from control group (p<0.05)
Chivapat S. et al. / Thai J. Vet. Med. 2010. 40(4): 45-53.
51 lymphoid tissue proliferation in the lung than their corresponding control groups. The satellite group of both sexes had significantly higher incidence of centrilobular hydropic degeneration in the liver tissue than their corresponding control group. Histopathological findings of the heart, kidney and intestine in all MPE-treated groups did not differ from those in the control group (Table 7 and 8). In addition, there was no remarkable lesion in other examined organs between the MPE-treated and control group.
group. Both BUN and creatinine in the group receiving MPE at dose of 500 mg/kg/day onward and those in the satellite group were significantly higher than those in the control group (Table 6). Effects of MPE on histopathological alterations: At autopsy, there was no remarkable macroscopic lesions in any organs of both MPE-treated and control groups. Histopathology of visceral organs revealed that the highest dose male and female groups had significantly lower incidence of bronchiole-associated
Table 7 Histopathological results of male and female rats receiving MPE for 6 months Organs Lung Heart Liver
Kidney Small intestine Large intestine
Microscopic findings BALT proliferation Myocardiosis Centrilobular hydropic degeneration Dilated tubule Hydronephrosis GALT proliferation in submucosa GALT proliferation in submucosa
Male rats Dose of MPE administered (mg/kg /day) Control 10 100 500 1000 1000-S
Female rats Dose of MPEadministered (mg/kg/day) Control 10 100 500 1000 1000-S
8/15 1/15 2/15
9/15 1/15 0/15
7/15 0/15 0/15
6/15 0/15 0/15
2/15* 0/15 6/15
0/15 NRL 1/15
0/15 NRL 3/15
0/15 NRL 2/15
0/15 NRL 3/15
1/15 NRL 1/15
4/15* NRL 1/15
NRL 0/15 1/15
NRL 0/15 3/15
NRL 1/15 1/15
NRL 4/15* 2/15
NRL 0/15 1/15
NRL 0/15 1/15
The results were expressed as the number of rats with pathological findings per total number of rats treated, 1000-S: the satellite group * significantly different from control group (p<0.05) (NRL: No remarkable lesions, BALT: Bronchiole-associated lymphoid tissue, GALT: Gut-associated lymphoid tissue)
Discussion In this study, an administration of MPE at any tested doses did not cause any overt toxic signs and mortality in the rats. The measurement of body weight indicated that MPE may depress the growth of the animals and the male rats were more susceptible to this effect than the female rats. Our result was different from a previous study by Towatana et al. (2010) saying that the oral administration of the 50% ethanolic extract in Wistar rats for three months did not affect the body weight at any time-points. This discrepancy may be caused by the difference in chemical constituents and their contents between the 95% and 50% ethanolic extract. Peaslee and Einhellig (1973) demonstrated that mice fed with diet containing tannic acid had retarded growth. In addition, the weanling rats receiving high tannin varieties of sorghum had significantly lower growth than those treated with low tannin varieties (Jambunathan and Mertz, 1973). Therefore, the result of poorer body weight in the male group treated with 500 mg/kg MPE and that in the highest dose of both sexes might partially be due to the effects of tannins. The significantly less food intake were observed at only three time-points in the male rats of highest dose group and only one time-point in the female receiving this dose. However, measurement of the weekly food intake at any other time-points in both groups did not show any discrepancies. Thus, it could not be stated that MPE suppressed the food intake of the animals. Almost all of the organs of the male rats of the highest dose and satellite groups as well as several organs of the male rats treated with 500 mg/kg/day
MPE revealed higher relative weights. These findings may be caused by the lower body weight. As histopathological results of such organs did not show any associated abnormalities. In addition, their actual weights did not reveal any changes when compared to the control group. Similar reasons may account for the increased relative weight of some organ of the female rats of the highest dose and satellite groups. Hematological analysis of blood samples from the female rats revealed no significant difference in the total WBC number between the highest dose and their control group. Thus, the increase of such parameter in only the satellite female group could due to biological variations among rats rather than the results of the extract. A decrease of eosinophils in both male and female groups receiving MPE at the doses of 500 and 1000 mg/kg/day as well as that in the satellite groups was likely due to the extract; however, this alteration was within normal range (Gad, 1992) and revealed no clinical significance. Neutrophil counts of the male rats receiving the highest dose was significantly higher and seemed to be dose-related. While Jejun et al. (2008) found that the same dose of 95% ethanolic extract of mangosteen did not cause any significant difference in hematological parameters including neutrophil in the male Sprague-Dawley rats. This discrepancies may be due to the difference in rat strains and duration of extract treatment. However, in this study, the alteration of neutrophils was slightly higher than the normal range (Gad, 1992) and the withdrawal of treatment could lead to reversibility of this effect. Clinical chemistry examination revealed the increase of ALT levels in the male rats receiving MPE at the
52 doses of 500 and 1000 mg/kg/day and also that of AST in the male and female rats receiving highest dose, which may be caused by MPE. Pramyothin et al. (2003) demonstrated that xanthones, the major compound isolated from mangosteen pericarp, caused the increase of both transaminases enzymes in isolated rat hepatocytes, which indicated the hepatoxic effect. In addition, mangostin has been shown to induce the significant increase of AST and ALT in rats treated when given by intraperitoneal or oral administration (Sornprasit et al., 1987). The decrease of total protein in the male rats receiving highest dose was slightly lower than the normal range (Pimainog et al., 2003) and this change could return to normal after the extract withdrawal. Possible causes of this finding may be decreased protein synthesis caused by hepatic insufficiency and/or increased protein catabolism (Stockham and Scott, 2002). Marzo et al. (2002) reported that chicken fed with tannic acid added diet exhibited a marked increase in the activities of liver cathepsin A and D which suggested the increase in protein catabolism. Thus, the tannin in MPE, at least in part, account for this finding. The increase of total bilirubin in the female rats receiving the highest dose was within normal range (0.00-0.55 mg/dl) as reported by Gad (1992). The decrease of glucose levels in the males and females receiving highest dose might contribute to the decreased gluconeogenesis in the liver, according to the hepatic insufficiency as shown by the increased hepatic enzyme activities (Stockham and Scott, 2002). The decrease of uric acid level in the male rats tended to be dose-related, however that in the highest dose group was within normal range (Gad, 1992). The decrease of cholesterol levels in the male rats receiving the extract at 500 mg/kg onward as well as the male satellite group might be due to the decreased production in the impaired hepatocytes (Stockham and Scott, 2002); nevertheless this alteration was within the reference values of the male rats (Pimainog et al., 2003). The increase of BUN and creatinine in the female rats treated with 500 and 1000 mg/kg/day as well as the increase of BUN in the male rats of the highest dose group were higher than the reference interval of male and female Wistar rats as reported by Pimainog et al. (2003). These alterations suggested that MPE might affect the kidney function and this effect was also observed in both sexes after the extract withdrawal. Histopathological examination of the lungs from the male and female rats of the highest dose group revealed a decreased incidence of bronchioleassociated lymphoid tissue proliferation. This finding might be due to the anti-inflammatory effect of the extract. Nakatani et al. (2002) demonstrated that γmangostin, a xanthone compound in the mangosteen fruit hull, inhibited the syntheses of prostaglandin E2 and enzymes cyclooxygenase I and II. A significantly higher incidence of centrilobular hydropic degeneration of the liver of the male rats of the satellite group, together with the increase AST levels may be indicative of hepatotoxic effects of the extract. While the female rats of the satellite group had an increase incidence of such histological alteration
Chivapat S. et al. / Thai J Vet Med. 2011. 41(1): 45-53. without any elevations of their AST or ALT enzymes. The reasons for this finding may be explained that the magnitude of the serum enzyme activity is not a reliable indicator regarding the type or degree of tissue injury (Lassen, 2004). Our results also suggested that the withdrawal of MPE treatment could not lead to reversibility of hepatotoxic effects within two weeks. Although the males of the satellite group had the higher incidence of the kidney tubular cyst, this alteration did not show any significant difference between the male rats of the highest dose and their control group. Furthermore, the female rats of the satellite and the highest dose group did not possess this alteration in their kidneys. Thus, this finding may not be concluded to be the results of MPE and needs to be further investigated. The increase of hydronephrosis incidence in the female rats receiving the extract at dose of 500 mg/kg/day could not be due to MPE since this was not dose dependent. Moreover, this change is considered incidental and thought to be congenital and inherited in many strains of rats (King and Russel, 2006). Other histopathological findings in the treatment groups did not show any dose dependency or any significant difference; therefore it could not be due to MPE. In conclusion, the six-month oral administration of MPE in Wistar rats at the doses of 10, 100, 500 and 1000 mg/kg/day revealed that MPE did not produce any overt pharmacotoxic signs and abnormalities in hematological values. However, MPE at dose of 500 mg/kg/day onward affected the body weight and produced the increase in ALT, BUN and creatinine in the tested animals. The highest dose MPE caused the significant increase in AST. In addition, the finding of hepatocellular degeneration after the highest dose withdrawal may be suggestive of the persistence in liver pathology caused by the highest dose extract. Hence, this study revealed that MPE at dose 500 mg/kg onward affected liver and kidney, and it could not be suggested that MPE are safe for the long term usage. Additional assessment of the appropriate dose range including further studies on hepatotoxicity of various chemical components in MPE are suggested to be investigated, which may be useful for the safe assessment and for the health product development from mangosteen pericarp extract.
Acknowledgement The authors would like to thank staffs of the Laboratory Animal Center of Department of Medical Sciences for animal facilities. We also thank Mr. Pornchai Sincharoenpokai for his technical assistance. This study was supported by grants from The Department of Medical Sciences 2008.
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53 prostaglandin E2 synthesis by mangosteen, a Thai Medicinal Plant. Biol Pharm Bull. 25(9): 1137-1141. Peaslee, M.H. and Einhellig, F.A. 1973. Tanic acidinduced alterations in mouse growth and pituitary melanocyte-stimulating hormone activity. Toxicol Appl Pharmacol. 25(4): 507-514. Pedraza-Chaverri, J., Cárdenas-Rodríguez, N., Orozco-Ibarra, M. and Pérez-Rojas, J.M. 2008. Medicinal properties of mangosteen (Garcinia mangostana). Food Chem Toxicol. 46: 3227-3239. Pimainog, Y., Yothinarak, A. and Jornrakate, P. 2003. Reference ranges for hematological and clinical chemistry values in Wistar rats. Bull Dept Med Sci. 45(1): 27-36. Pramyothin, P., Sapwarobol, S. and Ruangrungsi, N. 2003. Hepatoxic effects of xanthones extracted from rind of Garcinia mangostana in isolated rat hepatocytes. Thai J Pharm Sci. 27(3-4): 123-129. Reanmongkol, W. and Wattanapiromsakul, C. 2008. Evaluation of the analgesic, antipyretic and antiinflammatory activities of the extracts from the pericarp of Garcinia mangostana Linn. in experimental animals. Songklanakarin J Sci Technol. 30(6): 739-745. Sornprasit, A., Sripiyarattanakul, K., Chuay-Yim, P. and Tanakittithum, P. 1987. Preliminary toxicological study of mangosteen Songklanakarin J Sci Technol. 9(1): 51-57. Stockham, S.L. and Scott, M.A. 2002. Fundamental of Veterinary Clinical Pathology. Ames: Iowa State Press. 610 pp. Suksamrarn, S., Suwannapoch, N., Phakhodee, W., Thanuhiranlert, J., Ratananukul, P., Chimnoi, N. and Suksamrarn, A. 2003. Antimycobacterial activity of phenylated xanthones from the fruits of Garcinia mangostana. Chem Pharm Bull. 51(7): 857-859. Suksamrarn, S., Komutiban, O., Ratananukul, P., Chimnoi, N., Lartpornmatulee, N. and Suksamrarn, A. 2006. Cytotoxic prenylated xanthones from the young fruit of Garcinia mangostana. Chem Pharm Bull. 54: 301–305. Towatana, N.H., Reanmongkol, W., Wattanapiromsakul, C. and Bunkrongcheap, R. 2010. Acute and subchronic toxicity evaluation of the hydroethanolic extract of mangosteen pericparp. J Med Plant Res. 4(10): 969-974. Voravuthikunchai, S.P. and Kitpipit, L. 2005. Activity of medicinal plant extracts against hospital isolates of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect. 11: 510–512. Weecharangsan, W., Opanasopit, P., Sukma, M., Ngawhirunpat, T., Sotanaphun, U. and Siripong, P. 2006. Antioxidative and neuroprotective activities of extracts from the fruit hull of mangosteen (Garcinia mangostana Linn.). Med Princ Pract. 15: 281-287.
Genetic Characterization of Porcine Epidemic Diarrhea Virus (PEDV) Isolates from Southern Vietnam during 2009-2010 Outbreaks Do Tien Duy1, 4 Nguyen Tat Toan2 Suphasawatt Puranaveja3 Roongroje Thanawongnuwech1*
Abstract Porcine epidemic diarrhea virus (PEDV) spike (S) glyprotein and membrane (M) glycoprotein genes are believed to have genetic variation. The heterogeneity in those genomic sequences has been reported and is known essentially for the diverse PEDV pathogenicity. Eight southern Vietnamese PEDVs collected from severe watery diarrhea piglets of the recent emerging outbreaks (2009-2010) were sequenced and analyzed. The results revealed high nucleotide homology of the partial S gene of the current isolates at 98.9-100% and 99.7-100% identity of the full M gene among these isolates despite dividing into two subclusters of different provincial origins. It should be noted that the Vietnamese PEDVs contained high differences on nucleotide sequence of partial S gene with other reference isolates in Europe (Br1/87, CV777) and in Korea (Spk1, Chinju99, DR13 and KNU-0801). The phylogenetic relationship of both partial S and M protein genes indicated that the current Vietnamese PEDVs were in the same cluster with the Chinese isolates (JS-2004-2 and DX), the Thai isolates (07NP01, 08NP02 and 08CB01) and the recent Korean isolates (KNU-0802 and CPF299). The results suggested that the current Vietnamese PEDV isolates might have originated from the same Chinese ancestor undergoing genetic variation and possibly forming a new PEDV genotype in Vietnam. Keywords: phylogenetic analysis, pigs, porcine epidemic diarrhea virus, Vietnam 1Department
of Veterinary Pathology, Faculty of Veterinary Sciences, Chulalongkorn University, Henri-Dunant Rd, Pathumwan, Bangkok 10330, Thailand 2Department of Veterinary Internal Medicine and Pharmacology, Faculty of Animal Science and Veterinary Medicine, Nonglam University, Hochiminh city, Vietnam 3Veterinary Diagnostic Laboratory, Faculty of Veterinary Sciences, Chulalongkorn University, Henri-Dunant Rd, Pathumwan, Bangkok 10330, Thailand 4Department of Veterinary Microbiology and Infectious Diseases, Faculty of Animal Science and Veterinary Medicine, Nonglam University, Hochiminh city, Vietnam *Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 55-64.
Do Tien Duy et al. / Thai J Vet Med. 2011. 41(1): 55-64.
บทคัดย่อ ลักษณะทางพันธุกรรมของไวรัสพีอีดที ี่แยกได้จากภาคใต้ของประเทศเวียดนามระหว่างการระบาด ในปี ค.ศ. 2009-2010 โด เทียน ดุย1, 4 เหงียน ทัต โธน2 ศุภสวัสดิ์ บูรณเวช3 รุ่งโรจน์ ธนาวงษ์นุเวช1* ยีนของโปรตีนสไปค์และโปรตีนเมมเบรนของไวรัสพีอีดีเป็นยีนที่มีความหลากหลายทางพันธุกรรมซึ่งจะมีความแตกต่างกันในแต่ละ พื้นที่ มีรายงานว่า ลําดับทางพันธุกรรมของจีโนมที่ต่างกันในยีนดังกล่าวมีความเกี่ยวข้องกับพยาธิกําเนิดของโรค เมื่อทําการถอดรหัสและ วิเคราะห์พันธุกรรมของไวรัสพีอีดีจํานวน 8 ตัวอย่าง ซึ่งแยกได้จากลูกสุกรในฟาร์มทางใต้ของประเทศเวียดนามที่แสดงอาการท้องเสียในช่วง ที่มีการระบาดของโรคพีอีดีระหว่าง ปี ค.ศ. 2009-2010 พบว่าบางส่วนของยีนสไปค์และทั้งหมดของยีนเมมเบรนของไวรัสพีอีดีดังกล่าว มี ความเหมือนกันร้อยละ 98.9-100 และ 99.7-100 ตามลําดับ โดยสามารถแบ่งออกเป็น 2 กลุ่มย่อยตามจังหวัดที่พบเชื้อซึ่งมีความแตกต่าง จากไวรัสพีอีดีที่พบในทวีปยุโรป (Br1/87 และ CV777) และประเทศเกาหลี (spk1, Chinju99, DR13 และ KNU-0801) ผลการวิเคราะห์ ความสัมพันธ์ของลําดับนิวคลีโอไทด์ของเชื้อแบบแผนภูมิต้นไม้พบว่าเชื้อที่แยกได้จากการศึกษานี้จัดอยู่ในกลุ่มเดียวกับเชื้อไวรัสที่ก่อโรค ปัจจุบันในประเทศจีน (JS-2004-2 และ DX) ประเทศไทย (07NP01, 08NP02 และ08CB01) และประเทศเกาหลี (KNU-0802 และ CPF299) จากผลการศึกษาพบว่า ไวรัสพีอีดีที่ระบาดในประเทศเวียดนาม มีความเป็นไปได้ที่จะมีสายวิวัฒนาการมาจากไวรัสในประเทศจีน โดยมีการเปลี่ยนแปลงลักษณะทางพันธุกรรมดังกล่าวอาจเกิดขึ้นหลังจากระบาดมาระยะเวลาหนึ่ง จนพัฒนาเป็นไวรัสพีอีดีสายพันธุ์ใหม่ใน ประเทศเวียดนาม คําสําคัญ: phylogenetic analysis สุกร porcine epidemic diarrhea virus เวียดนาม 1 ภาควิชาพยาธิวทิ ยา คณะสัตวแพทยศาสตร์ จุฬาลงกรณ์มหาวิทยาลัย ปทุมวัน กรุงเทพฯ 10330 2 Department of Veterinary Internal Medicine and Pharmacology, Faculty of Animal Science and Veterinary Medicine, Nonglam University, Hochiminh city, Vietnam 3 หน่วยชันสูตรโรคสัตว์ คณะสัตวแพทยศาสตร์ จุฬาลงกรณ์มหาวิทยาลัย ปทุมวัน กรุงเทพฯ 10330 4 Department of Veterinary Microbiology and Infectious Diseases, Faculty of Animal Science and Veterinary Medicine, Nonglam University, Hochiminh city, Vietnam *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction Porcine epidemic diarrhea (PED) is a contagious disease caused by a Coronavirus called porcine epidemic diarrhea virus (PEDV) producing acute enteritis and fatal watery diarrhea with high mortality particularly in suckling pigs up to 90% (Pensaert and Yeo, 2006). PED was first identified in England in 1971 and has currently become a problematic disease causing massive economic losses in many countries, mainly, in Europe and Asia (Pensaert and Yeo, 2006; Park et al., 2007b; Chen et al., 2008; Puranaveja et al., 2009). Heterogeneity in genomic sequences has been reported and is known essentially as the cause for the diverse PED pathogenicity. A number of molecular investigations have been performed and revealed low to high variation of nucleotide sequences of PEDV, especially
in the S glycoprotein gene (Duarte and Laude., 1994; Kocherhans et al., 2001; Yeo et al., 2003; Kang et al., 2005; Park et al., 2007b; Puranaveja et al., 2009; Lee et al., 2010). Phylogenetic analysis comparing PEDVs from various countries demonstrated that PEDVs were classified into three distinct groups based on nucleotide homology of the partial S gene and M gene (Park et al., 2007b; Chen et al., 2008; Puranaveja et al., 2009; Lee et al., 2010). In early 2009, emerging PED outbreaks confirmed by pathological features and RT-PCR in most of the southern provinces of Vietnam caused massive economic losses in the swine industry (unpublished data). The disease investigation revealed that acute diarrhea syndrome occurred in all age groups of pigs. The affected animals manifested acute watery diarrhea condition and finally recovered mostly in adult animals. Suckling piglets suffered from severe watery diarrhea, dehydration and died
Do Tien Duy et al./ Thai J Vet Med. 2011. 41(1): 53-62. within a few days. Morbidity in suckling pigs reached 100% and mortality among provinces ranged from 65% to 91%. Therefore, the objective of this study was to genetically characterize the current PEDV isolates for elucidation of the epidemiologic relationship among PEDV isolates during the 2009-2010 outbreaks in southern Vietnam.
Materials and Methods Sample collection: Total 8 PEDV isolates taken from 3-10 day-old piglets suffering from watery diarrhea and dehydration on five different affected farms of three provinces in southern Vietnam were included in this study. RNA isolation: Small intestinal samples were homogenized by adding PBS to 10% suspension, centrifuged at 3000 rpm for 10-15 min at 4oC. The supernatant of 1-3 ml was collected into the sterile centrifuged tubes using for total RNA isolation according to the protocol of the commercial kit’s instruction (Promega, Madison, WI, USA). RT-PCR: Two different genomic regions were amplified, 651 bp fragment of partial S glycoprotein gene and 715 bp fragment of full M gene, by using the two pairs of primers based on previous publication (Park et al., 2007b; Chen et al., 2008). Briefly, nucleotide strands of the partial S gene primers are 5’TTCTGAGTCACGAACAGCCA-3’ (PS1, forward), 5’CATATGCAGCCTGCTCTGAA-3’ (PS2, backward) and of M gene primers, 5’-CCCCAGTACTGTTA TTGACGTATAAAC-3’ (PM1, forward), 5’-GTTTAG ACTAAATGAAGCACTTTC-3’ (PM2, backward),
57 respectively. One tube RT-PCR reaction was used to amplify partial S and M glycoprotein genes of PEDV (AccessQuickTM, Promega, Madison, USA). Exactly, 4 µl RNA template was mixed with a reaction mixture, which contained 10 µl of 2x AccessQuickTM Master Mix (Promega, Madison, WI, USA), 1 µl of each specific primer (10 µM), 0.5 µl of MgCl2 (25 µM), 0.5 µl and AMV reverse transcriptase (10 u/µl). Then, 8 µl nuclease-free water was added to reaching the total volume reaction of 25 µl. The RT-PCR reactions were run in a Thermal hybrid PCR machine (USA), divided into three stages. Firstly, reverse transcription reaction incubated at 48oC for 45 min to make the first strand cDNA synthesis. Then, the second strand cDNA synthesis and PCR amplification were denatured at 95oC for 2 min (01 cycle), repeatedly denatured at 94oC for 30 sec for 30 cycles, annealed at 53oC for 60 sec and extended at 72oC for 60 sec. Additional step is the final extension at 72oC for 5 min. The last stage is to hold the PCR products at 4oC. Analyzing the PCR products by agarose gel electrophoresis of 1.5% was readily visible by UV transillumination of an ethidium bromide-stained gel. Sequencing: Purified PCR products corresponding to the partial S glycoprotein gene and full M gene were sequenced by 1st BASE Pte Ltd (Singapore). All sequencing reactions were carried out in duplicate and all sequences was determined by sequencing both strands (forward and backward strands). The sequencing process used BigDye Terminator v3.1 cycle sequencing kit performed in 96-well plate (BigDye® Terminator v3.1 Cycle Sequencing Kit’s protocol).
Table 1 Sources of viruses Order Isolates Countries and year of sampling Accession number References 1 CV777 S/M Belgium, 1977 AF353511.1 Kocherhans et al., 2001 2 Br1/87S/M Britain, 1987 Z25483/Z24733.1 Duarte and Laude, 1994 China, 2004 AY653204.1/ Y653205.1 Unpublished 3 JS-2004-2 S/M China, 2006 EF185992.1 Unpublished 4 LZC M China, 2007 EU031893.1 Unpublished 5 DX S/M China, 2006 EU033965.1 Chen et al., 2010 6 CHIMT06 M Korea, 2002 AF500215.1 Kang et al., 2005 7 Spk1 S Korea, 1999 AY167585.1 Yeo et al., 2003 8 Chinju99 S/M Korea, 2006 DQ462404.2 Park et al., 2007a 9 DR13 S Korea, 2008 GU180142.1 Lee et al., 2010 10 KNU-0801 S Korea, 2008 GU180143.1 Lee et al., 2010 11 KNU-0802 S Korea, 2003 FJ687455.1 Unpublished 12 M1763 M Korea, 2007 FJ687467.1 Unpublished 13 CPF299 M Korea, 1997 AF015888.1 Kweon et al., 1999 14 KPEDV-09 M Thailand, 2007 FJ196196.1 Puranaveja et al., 2009 15 07NP01 S/M Thailand, 2008 FJ196204.1 Puranaveja et al., 2009 16 08NP02 S/M Thailand, 2008 FJ196197.1 Puranaveja et al., 2009 17 08CB01 S Thailand, 2008 FJ196194.1 Puranaveja et al., 2009 18 KU06RB08 M Vietnam, 2009 HQ883485/HQ883479 In this study 19 VN92 S/M In this study Vietnam, 2009 HQ883486 20 VN94 S In this study 21 VN97 S Vietnam, 2010 HQ883487 In this study 22 VN103 S/M Vietnam, 2010 HQ883488/HQ883480 In this study 23 VN109 S/M Vietnam, 2010 HQ883489/HQ883481 In this study 24 VN112 S/M Vietnam, 2010 HQ883490/HQ883482 In this study 25 VN116 S/M Vietnam, 2010 HQ883491/HQ883483 In this study 26 VN122 S/M Vietnam, 2010 HQ883492/HQ883484 SIsolate used for sequence analysis of the partial S gene; MIsolate used for sequence analysis of the full M gene
Sequencing analysis: Nucleotide sequences of the current Vietnamese PEDV isolates and other selected isolates presented on Table 1 (Genbank: http://www.ncbi.nlm.nih.gov) were edited, aligned
and analyzed with Chromas 2.33, Bioedit v126.96.36.199, and ClustalX 2.0.11 program. The phylogenetic trees and deduced amino acid sequences were then generated based on the Maximum likelihood method (Saitou
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and Nei, 1993) with the MEGA 5.0. The relative support for each branch and the bootstrap value of 1000 replicates were computed (Tamura et al., 2007).
Results Eight southern Vietnamese PEDVs were collected from recent PEDV-affected commercial swine farms (2009-2010) in three different provinces. These isolates were confirmed by one-step RT-PCR amplification to recognize the specific products of partial S gene and full M gene. They were sequenced and named VN92S1, VN94S2, VN97S3, VN103S4, VN109S5, VN112S6, VN116S7, VN122S8 for partial S gene analysis and VN92M1, VN9103M2, VN109M3, VN112M4, VN116M5, VN122M6 for full M gene analysis. Sequence homology Partial S gene: The pairwise alignment of the southern Vietnamese PEDV isolates showed high nucleotide homology to each other (98.9-100%). VN92S1 had minor different nucleotide and encoded amino acid sequence with other isolates (VN94S2, VN97S3, VN103S4, VN109S5, VN112S6, VN116S7, VN122S8) at 98.9% and 97.3% identity, respectively. It should be noted that the current PEDV isolates contained variable differences on nucleotide sequences of the partial S gene with other reference isolates (Table 2) sharing 95.6-96.4% nucleotide identity with the European prototypes (CV777, Br1/87) and 91.4–97.8% identity with the Korean isolates (Chinju99, Spk1, DR13, KNU-0801, KNU0802). Interestingly, the Vietnamese isolates shared high nucleotide homology with the Chinese isolates (JS-2004-2, DX) and the Thai isolates (07NP01, 08NP02, 08CB01) at 97.7–98.5% and 98.8–99.5%, respectively. The average of nucleotide sequence identity of the current Vietnamese PEDVs revealed high correlation together with those of neighboring countries such as the Chinese isolate (JS-2004-2) at
98.4%, the Thai isolates at 99.35% and the recent Korean isolate (KNU-0802) at 97.51% (Table 3). For deduced amino acid sequence analyses of the partial S glycoprotein gene, the current Vietnamese PEDV isolates also showed high amino acid sequence homology ranging from 97.3-100% and were closely related to the Chinese isolates (JS-2004-2, DX) and the Thai isolates (07NP01, 08NP02, 08CB01) approximately 92.3-95.2% and 97.3-99.3% identity, respectively. In contrast, these current Vietnamese isolates had quite a lot of differences in genetic distance of amino acid sequences with those in Europe (CV777, Br1/87) and in Korea (Chinju99, Spk1, DR13, KNU-0801) at 85.6-87.9% and 80.0-93.8%. Likewise, the average percentages of peptide sequence homology of the Vietnamese PEDVs shared high relationship with the mentioned PEDVs in the neighboring countries including China, Korea and Thailand (Table 3) at 94.3%, 92.87% and 98.81%, respectively. Full M gene: Homology of nucleotide and deduced amino acid sequences in full M gene of the current Vietnamese PEDV isolates shared considerably high homology (99.7-100% and 99.0-100%) to each other (Table 4). Similar to the partial S gene, the nucleotide and amino acid sequence homology were computed to make clearly the relationship among groups and within each group. Group 1 shared 97.89% and 96.18% in nucleotide and deduced peptide sequence identity, respectively, with group 2 and 97.33% and 95.09% with group 3 (Fig. 4). Within group 1, the current Vietnamese PEDV isolates shared 98.89% and 97.07% of nucleotide and amino acid sequence identity with the Thai isolates (07NP01, 08NP02, KU06RB08), 98.53% and 97.49% identity with the Chinese isolates (JS-2004-2, DX), and 98.56% and 96.71% identity with the recent Korean isolates (CPF299, M1763). Furthermore, the Thai isolates shared 98.24% and 96.97% of nucleotide and peptide
Table 2 Comparison between percent of homology of nucleotide and encoded amino acid sequences of the partial S gene of the southern Vietnamese PEDV isolates
Nucleotide identity (%) in lower triangle of table, Decoded amino acid identity (%) in upper triangle of table
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Table 3 Comparison percent genetic distance (average % identity) of nucleotide/deduced amino acid sequences of the partial S gene Group 1 Group 2 Europe Korea China Thailand Vietnam Europe 97,21 96,85 96,40 96,24 Korea 90,11 98,10 97,80 97,51 Group 1 96,20 China 90,85 95,19 98,85 98,18 Thailand 87,10 93,41 95,54 99,35 Vietnam 87,31 92,87 94,30 98,81 Group 2 91,58 Group 3 82,59 90,90 Nucleotide identity (%) in upper triangle of table, Decoded amino acid identity (%) in lower triangle of table PEDV strains
sequence homology with the Chinese isolates, 98.95% and 97.74% identity with the recent Korean isolates. The Chinese and the recent Korean isolates shared 98.06% and 96.35% of nucleotide and decoded
aminoacid sequence identity, respectively. In particular, the Thai isolates had absolute homology of M gene with the recent Korean isolate (CPF299) at 100% identity. Similarly, the current Vietnamese
Figure 1 Comparison of encoded peptide sequence alignment of the partial S gene of eight Vietnamese PEDV isolates (VN92S1, VN94S2, VN97S3, VN103S4, VN109S5, VN112S6, VN116S7, VN122S8) and selected reference PEDV strains (European strains, CV777; Chinese isolates, JS-2004-2 and DX; Korean isolates, Spk1, KNU-0801, KNU-0802; Thai isolates, 07NP01, 08NP02). Dash (.) reveals the amino acid identity of isolates compare with prototype isolate (CV777). There were some sites in sequence with different encoded amino acids due to substitution of amino acids comparing with CV777.
Do Tien Duy et al. / Thai J. Vet. Med. 2011. 41(1): 53-62.
Figure 2 Comparison of deduced amino acids sequence of the full M gene of six Vietnamese PEDV isolates (VN92M1, VN103M2, VN109M3, VN112M4, VN116M5, VN122M6) and selected reference PEDV strains (European strains, CV777 and Br1/87; Chinese isolates, JS-2004-2, LZC, DX and CHIMT06; Korean isolates, Chinju99, M1763, CPF299, KPEDV-09); Thai isolates, 07NP01, 08NP02 and KU06RB08). Dash (.) reveals the amino acid identity of isolates compare with prototype isolate (CV777). There were some sites in sequence with different amino acids due to substitution of amino acids comparing with CV777.
isolates had the closest relation with JS-2004-2 at 99.7% and 99.0% of nucleotide and decoded amino acid sequence identity, respectively. Genetic characterization Partial S gene: The nucleotide sequences of partial S gene of the southern Vietnamese PEDV isolates were composed of 600 nucleotides (encoded approximately 200 amino acids), located from position 1530 to 2130 in full length of S gene responding for epitope determinant of virus (data not shown) in which the nucleotide sequence revealed high variable sites from 1561 to 1600 and 1821 to 1851 (520 to 533 and 598 to 617 residues of amino acid sequence, respectively). There was a total 15 amino acids changes within 25 nucleotides substitution corresponding to the partial S gene (Fig 1).
Full M gene: The nucleotide sequence of the full M gene of the current Vietnamese PEDV isolates were composed of 687 nucleotides encoded approximately 225 amino acids. The six current Vietnamese PEDV isolates and the European prototype (CV777) revealed high homology containing 13 nucleotide changes corresponding to 2 residues substitution on amino acid sequences. Likewise, deduced amino acid of M gene shared extremely high homology among PEDV isolates of the neighboring countries of Asia (Fig. 2). Phylogenetic analysis Partial S gene: Phylogenetic analysis based on nucleotide and encoded amino acid sequences of the partial S gene revealed PEDV isolates divided into three groups (Fig. 3). Group 1 contained most of PEDV isolates in suffered countries including Europe (CV777, Br1/87), China (JS-2004, DX), Korea (DR13,
Do Tien Duy et al./ Thai J Vet Med. 2011. 41(1): 53-62.
Figure 3 Dendrogram based nucleotide sequence of the partial S gene among eight southern Vietnamese PEDV isolates (VN92S1, VN94S2, VN97S3, VN103S4, VN109S5, VN112S6, VN116S7, VN122S8) with those of other reference strains (European strains, CV777 and Br1/87; Chinese isolates, JS-20042 and DX; Korean isolates, Chinju99, DR13, Spk1, KNU-0801, KNU-0802; Thai isolates, 07NP01, 08NP02, and 08CB01). Multiple alignment method performed by using ClustalX program. The scale bars indicate the number of 0.01 estimate evolutionary time. KNU-0802), Thailand (07NP01, 08NP02, 08CB01) and Vietnam (in this study). Group 2 comprised Spk1, KNU-0801 (Korean isolates). Group 3 comprised previous Korean isolate (Chinju99). The current Vietnamese PEDV isolates dropped into a distinct clade compared with other reference isolates. They shared high homology. However, at least two separated clades in which clade 1 comprising VN94S2, VN97S3, VN103S4, VN109S5, VN112S6, VN116S7, VN122S8 and clade 2 containing only VN92S1 with 98.9% sequence identity were observed. It should be noted that the Vietnamese isolates contained high differences on the nucleotide sequence of the partial S gene with other prototypes in Europe (CV777, Br1/87) and in Korea (Chinju99, Spk1, KNU0801). However, the current Vietnamese isolates, the Thai isolates, the Chinese isolates and the recent Korean isolate (KNU-0802) were clustered into the same subgroup on the phylogenetic tree.
Figure 4 Dendrogram based on nucleotide sequence of the full M gene among six southern Vietnamese PEDV isolates (VN92M1, VN103M2, VN109M3, VN112M4, VN116M5, VN122M6) with those of other reference strains (European strains, CV777 and Br1/87; Chinese isolates, JS-2004-2, LZC, DX and CHIMT06; Korean isolates, Chinju99, M1763, CPF299; Thai isolates, 07NP01, 08NP02 and KU06RB08). Multiple alignment method performed by using ClustalX program. The scale bars indicate the number of 0.002 estimate evolutionary time.
Full M gene: PEDVs were similarly divided into three distinct genetic groups (Fig 4). Group 1 comprised the Vietnamese isolates (in this study), the Chinese isolate (JS-2004-2), the recent Korean isolates (CPF299, M1763) and the Thai isolates (07NP01, 08NP02, KU06RB08). The current Vietnamese PEDV isolates had close relationship with the mentioned above recent isolates from Asian countries including China (JS-2004-2), Thailand (07NP01, 08NP02, KU06RB08), and Korea (CPF299) clustering into a separated subgroup in phylogenetic tree. Group 2 comprised Chinju99 (Korea), DX and CHIMT06 (China). Group 3 included CV777, Br1/87 (Europe) and LZC (China).
Do Tien Duy et al. / Thai J Vet Med. 2011. 41(1): 55-64.
Table 4 Comparison between percent of homology of nucleotide and encoded amino acid sequences of full M gene of the southern Vietnamese PEDV isolates
Nucleotide identity (%) in lower triangle of table; Decoded amino acid identity (%) in upper triangle of table
Discussion Porcine epidemic diarrhea virus (PEDV) is classified into genetic group 1 of Coronaviruses. Spike (S) glycoprotein and membrane (M) glycoprotein genes are believed to have genetic variation geographically (Britton et al., 1991; Cavanagh et al., 1992; Adzhar et al., 1995; Ballesteros et al., 1997; Leparc-Goffart et al., 1997; Kingham et al., 2000; Saif, 2004; Weiss and Navas-Martin, 2005). Moreover, the heterogeneity in those genomic sequences has been reported and is known essentially for the diverse PEDV pathogenicity (Lai et al., 2007; Lee et al., 2010). PEDV genome is considered to have a diversity based on previous studies (Kweon et al., 1999; Kocherhans et al., 2001; Yeo et al., 2003; Jinghui and Yijing, 2004; Junwei et al., 2006; Park et al., 2007b; Li et al., 2009; Puranaveja et al., 2009; Lee et al., 2010). The genetic regions of nucleotide sequence containing the highest variations are C-terminal and N-terminal regions of S1domain which are used to analyze genetic relationship among PEDVs in this study (Park et al., 2007b; Lee et al., 2010). The M gene of Coronaviruses seems to be more conservable than the S gene (Lai et al., 2007; Chen et al., 2008; Puranaveja et al., 2009). The important role of M gene contributes to the assembly of process of viral nucleocapsid and membrane of internal structure as well as to the interferon secretion stimulation (Lai et al., 2007). The phylogenetic analysis showed the close relationship among isolates of neighboring countries in group 1 in China (JS-20042, DX), in Korea (KNU-0802, CPF299, and M1763), in Thailand (07NP01, 08NP02, 08CB01, KU06RB08) and in Vietnam. Particularly, Vietnamese PEDV isolates are extremely related to JS-2004-2 and the Thai isolates are absolutely related to the Korean isolate (CPF299) with 100% nucleotide and amino acid identity of M gene. These evidences suggests that the Chinese-like isolates have been responsible for the recent Asian outbreaks prevailing in Korea, Thailand and Vietnam (Park et al., 2007b; Puranaveja et al., 2009; Lee et al., 2010). Interestingly, geographic risk
factor seem to play a minor role in this PED outbreak similar to the outbreaks in Thailand and Korea since the outbreak occurred first in the southern part of Vietnam faraway from China. In addition, animal movement among those countries is limited. Other risk factors including poor bio-security application on fomites, animals and humans may play a major impact in the disease outbreak among those affected countries. Within the eight southern Vietnamese PEDV isolates, there was a small difference in VN92S1 compared with other Vietnamese isolates since it was isolated from a different farm and a different province (Farm 1, Binhduong). Two other isolates (VN94S4, VN97S3) collected from another farm in Binhduong also revealed minor differences in nucleotide and amino acid sequence with the other isolates. Therefore, the isolates from Binhduong were grouped into a distinct sub-cluster demonstrating that the current PEDV isolates have gradually had genetic diversity.
In conclusion, the phylogenetic analysis indicated that these current Vietnamese PEDV isolates have originated from the same Chinese ancestor and they were gradually undergoing genetic variation and forming a new PEDV sub-cluster in Vietnam. Furthermore, these results suggested that the Chinese isolates (JS-2004-2) could be the ancestor of the current PEDV outbreaks transmitting to the neighboring countries of Asia including Korea, Thailand and Vietnam (Park et al., 2007b; Chen et al., 2008; Puranaveja et al., 2009). It should be noted that the epidemiology of PED outbreaks is commonly related to geographic influence by transmission and circulation of the causative agent among neighboring countries. Since emerging in Europe, PED outbreaks have been worldwide spread to many geographical areas but not in the American continents excepting an old report of PED-induced outbreak in Canada (Turgeon et al., 1980). The reasons explained for the
Do Tien Duy et al./ Thai J Vet Med. 2011. 41(1): 53-62. absence of PED in Americas might be the geographical separation from the arising places of emerging virus and the availability of good preventive strategies in those countries. In addition, no animal movement from the PEDV endemic areas has been introduced into the North American continent. Normally, movement of live pigs, breeding stocks supplying for commercial farms and human movement among the countries could be major risk factors for the transmission of any emerging disease. In the current Vietnamese PED outbreaks, poor biosecurity application on fomites, animals and humans were considered main risk factors. Spreading among neighboring farms after the first introduction in the area is mostly due to animal and human movement as well as contaminated vehicles. Therefore, the clear elucidation of the origin and transmitted route of this emerging PEDV may contribute to a better understanding on the epidemiology and finally to effective prevention and control of the disease.
Acknowledgement The authors would like to thank “The Graduate Scholarship Program for Faculty Member for Neighboring Countries” for giving Mr. Do Tien Duy an opportunity to further his MSc study at the Veterinary Pathobiology program, Chulalongkorn University. We are also grateful to Dr. Nguyen Tat Toan, lecturer of Nonglam University, Vietnam and Mr. Keatipong Mongkolwit, officer of C.P. Vietnam Livestock Corporation for their assistance in collecting samples.
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Immunolocalization of Estrogen Receptor beta, Androgen Receptor and Ki-67 Protein in Testicular Tissues of Unilateral Cryptorchidism Boar Sukanya Manee-in* Pinthira Thiengthiantham Chanlika Prommapan Kampon Kaeoket
Abstract This study was performed to investigate histological structure, expressions of estrogen receptor beta (ERβ), androgen receptor (AR) and proliferation marker (Ki-67 protein) in scrotal and abdominal testicular tissues of unilateral cryptorchidism prepubertal boars. Testicular tissues were obtained from 8 unilateral cryptorchidism boars. Immunohistochemical staining for ERβ, AR and Ki-67 protein was performed by avidin-biotin-peroxidase complex (ABC) method. The similar histological structure of both scrotal and abdominal testicular tissues was observed. Immunolocalization of Ki-67 was found in the nuclei of germ cells, interstitial cells and Sertoli cells of both scrotal and abdominal testicular tissues. For ERβ and AR, the immunolocalization was found in germ cells and interstitial cells of both scrotal and abdominal testicular tissues. Based on the statistical analysis, the ERβ expression in the interstitial area of scrotal testis was significantly higher than in the abdominal testis. A tendency of more expressions of Ki-67 protein (p=0.40) in the seminiferous tubule of the scrotal testicular tissues than in the abdominal testicular tissues was found. The expressions of AR in scrotal and abdominal testicular tissues were not significantly different. In conclusion, the histological structures of scrotal and abdominal testis of unilateral cryptorchidism boars are not different. However, there are differences in their immunolocalization patterns between the scrotal and abdominal testis as well as seminiferous tubules and interstitial areas. Keywords: androgen receptor, cryptorchidism boar, estrogen receptor beta, Ki-67 Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Salaya, Putthamonthon, Nakhon Pathom 73170, Thailand Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 65-69.
Manee-in S. et al. / Thai J Vet Med. 2011. 41(1): 65-69.
บทคัดย่อ ตําแหน่งของตัวรับฮอร์โมนเอสโตรเจน ชนิดเบต้า ตัวรับฮอร์โมนแอนโดรเจน และโปรตีน Ki-67 ในเนื้อเยื่ออัณฑะของสุกรที่เป็นทองแดงข้างเดียว สุกัญญา มณีอินทร์* ปิณฑิรา เที่ยงเธียรธรรม ฌัลลิกา พรหมมาพันธุ์ กัมพล แก้วเกษ การศึกษาครั้งนี้มีวัตถุประสงค์ เพื่อศึกษาลักษณะทางจุลพยาธิวิทยา การแสดงออกของตัวรับฮอร์โมนเอสโตรเจนชนิดเบต้า ตัวรับ ฮอร์โมนแอนโดรเจน และโปรตีนงอกขยาย (โปรตีน Ki-67) ในเนื้อเยื่ออัณฑะที่อยู่ในถุงหุ้มอัณฑะ และในช่องท้องของสุกรก่อนวัยเจริญพันธุ์ที่ เป็นทองแดง เนื้อเยื่ออัณฑะถูกเก็บจากสุกรอายุ 2 เดือน จํานวน 8 ตัว ใช้วิธี avidin-biotin-peroxidase complex (ABC) ของทางอิมมูนโน ฮิสโตเคมีในการตรวจหาตัวรับฮอร์โมนเอสโตรเจนชนิดเบต้า ตัวรับฮอร์โมนแอนโดรเจน และโปรตีน Ki-67 ผลการศึกษา พบว่าลักษณะทาง จุลพยาธิวิทยาไม่มีความแตกต่างกันระหว่างเนื้อเยื่ออัณฑะที่อยู่ในถุงหุ้มอัณฑะและภายในช่องท้อง พบการมีอยู่ของโปรตีน Ki-67 ที่เซลล์ สืบพันธุ์ เซลล์ interstitial และเซลล์ Sertoli ของเนื้อเยื่ออัณฑะที่อยู่ในถุงหุ้มอัณฑะและภายในช่องท้อง และพบการมีอยู่ของตัวรับฮอร์โมน เอสโตรเจนชนิดเบต้า และตัวรับฮอร์โมนแอนโดรเจนที่เซลล์สืบพันธุ์ และเซลล์ interstitial ของอัณฑะที่อยู่ในถุงหุ้มอัณฑะและภายในช่อง ท้อง ผลการวิเคราะห์ทางสถิติพบการแสดงออกของตัวรับฮอร์โมนเอสโตรเจน ชนิดเบต้า ที่บริเวณ interstitial ของเนือ้ เยือ่ อัณฑะทีอ่ ยูภ่ ายใน ถุงหุ้มอัณฑะมากกว่าอัณฑะภายในช่องท้องอย่างมีนัยสําคัญ และพบแนวโน้มการแสดงออกของโปรตีน Ki-67 ใน seminiferous tubule ของเนื้อเยื่ออัณฑะที่อยู่ในถุงหุ้มอัณฑะมากกว่าเนื้อเยื่อที่อยู่ในช่องท้อง (p=0.40) จากการศึกษาไม่พบความแตกต่างระหว่างการแสดงออก ของตัวรับฮอร์โมนแอนโดรเจนของเนื้อเยื่ออัณฑะที่อยู่ในถุงหุ้มอัณฑะและภายในช่องท้อง จากผลการศึกษาสามารถสรุปได้ว่า ลักษณะทางจุล พยาธิวิทยาของเนื้อเยื่ออัณฑะที่อยู่ภายในถุงหุ้มอัณฑะและภายในช่องท้องไม่มีความแตกต่างกันในสุกรที่เป็นทองแดง อย่างไรก็ตาม พบความ แตกต่ า งของรู ป แบบการติ ด สี อิ ม มู น โนฮิ ส โตเคมี ร ะหว่ า งเนื้ อ เยื่ อ อั ณ ฑะที่ อ ยู่ ภ ายในถุ ง หุ้ ม อั ณ ฑะและภายในช่ อ งท้ อ ง และระหว่ า ง seminiferous tubule และบริเวณ interstitium คําสําคัญ: ตัวรับฮอร์โมนแอนโดรเจน สุกรที่เป็นทองแดง ตัวรับฮอร์โมนเอสโตรเจน ชนิดเบต้า Ki-67 ภาควิชาเวชศาสตร์คลินิก และการสาธารณสุข คณะสัตวแพทยศาสตร์ มหาวิทยาลัยมหิดล ศาลายา พุทธมณฑล นครปฐม 73170 *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction Cryptorchidism is a common reproductive defect, which is a failure of one testis (unilateral cryptorchidism) or both testes (bilateral cryptorchidism) to descend to the scrotum (Pinart et al., 1999b). The impairment of the contralateral scrotal testis may be caused by the malfunction of the cryptorchid testis (Pinart et al., 1999a; Pinart et al., 2002). It is well documented that estrogen is an important hormone for male reproductive function, in which it influence spermatogenesis and Sertoli-germ cells interaction. The physiological role of estrogen in males involve several steps of sperm production and maturation. Estrogen receptors are needed to exert estrogen action. The expression of estrogen receptor beta (ERβ) is found in immature boar testicular tissue, whereas the expression of estrogen receptor alpha (ERα) is observed in testes of mature boar (Rago et al., 2004).
Androgen plays an important role in male characteristics and accessory male sex organs development. Moreover, it plays role in the initiation of spermatogenesis (Wang et al., 2009). A previous study demonstrated that the presence of androgen receptor in boar testicular tissue was related to the expression of estrogen receptors (Ramesh et al., 2007). The presence of androgen receptor (AR) in Sertoli cells and Leydig cells are associated with various processes of spermatogenesis (Wang et al., 2009). Proliferation of several cell types in seminiferous tubules is involved in spermatogenesis. There are many cell types in testicular tissue that can be proliferated such as germ cells, Sertoli cells and Leydig cells. (Sriraman et al., 2000; Angelopoulou et al., 2008). Ki-67 is a well-established proliferation marker (Gerdes et al., 1991). A detailed in cell cycle analysis showed that the Ki-67 nuclear antigen was expressed in G1, S, G2, and mitosis, but not in resting cell in G0 phase (Gerdes et al., 1984).
Manee-in S. et al./ Thai J Vet Med. 2011. 41(1): 65-69. Until the present, the expressions of ERβ and AR in relation to proliferation activity in testicular tissues of cryptorchidism boars have not been fully investigated. In order to understand the role of steroid receptors; ERβ and AR presented in various cell types cryptorchidism testis and normal testis in connection with the initiation of spermatogenesis, the expressions of ERβ, AR and Ki-67 protein in scrotal and abdominal testicular tissues of the same boar are investigated.
Materials and Methods The experimental plan was approved by the Ethical Committee for Experimentation with Animals at Faculty of Veterinary Science, Mahidol University. Animals: Eight unilateral cryptorchidism boars aged 2 months old were investigated. The animals were kept in an open house, fed with commercial diet and given water ad libitum. Collection of samples: Both testes were collected from each boar by surgical excision under general anesthesia. Approximately 1x1 cm. of testicular tissue samples were fixed in 4% (w/v) paraformaldehyde for 48 hours. Thereafter they were dehydrated, embedded in paraffin and 4 µm thick sections were cut from each block and mounted on 3-aminopropyl triethoxysilane (Sigma-Aldrich, St Louis, MO) coated slides. The sections were kept until the immunohistochemical procedure was performed. Immunohistochemistry: The specimens were deparaffinized in xylene and rehydrated in graded ethanol. After washing with distilled water, they were subjected to high-temperature antigen retrieval by incubation with 0.01M citrate buffer, pH 6.0 in a microwave at high power (800W) for 5 min followed by long heating at 240W for 3 min and at 400W for 20 min (Manee-in and Srisuwatanasagul, 2011), at 800W for 30 min (5 min x 6) for ERβ and AR. The slides were rinsed with 10mM, pH 7.4 phosphate-buffered saline (PBS). The following procedures were performed at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min, then the tissues were rinsed with PBS. Non-specific staining was eliminated by incubating the sections with normal horse serum for 30 min. The sections were incubated with specific primary mouse monoclonal antibody to Ki-67 (clone MIB-1, dilution 1:100, Dako, Denmark) for 2 hours at room temperature, primary rabbit polyclonal antibody to ERβ (clone H-150, dilution 1:100, Santa Cruz Biotech, CA) for 3 hours at room temperature, and rabbit polyclonal antibody to AR (clone N-20,
67 dilution 1:100, Santa Cruz Biotech, CA) for all slides were placed in a humidified chamber. After primary antibody binding, the sections were washed in PBS and incubated with the secondary antibody in a dilution of 1:200 for 30 min. After the slides were washed in PBS the sections were incubated for 30 min with horseradish peroxidase avidin biotin complex (Vectastain® ABC kit, Vector Laboratories, Inc., USA). After the final wash with PBS, the color was developed with a freshly prepared solution of 3, 3’diaminobenzidine (DAB kit, Vector Laboratories, Inc., USA). All sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted with glycerine gelatin for investigation under a light microscope. Sections treated with non-immune serum, instead of the specific antibody, were used as negative controls. Sections from normal pig ovary, which is known to express ERβ, normal pig intestine, which is known to express Ki-67 protein, and normal testicular tissue from pubertal boar were served as positive controls. Evaluation of immunolabelling intensity: Each tissue section was acquired with a Nikon ECLIPSE TE2000U.The average labeling intensity percentage evaluated by image analysis (Image-Pro® PLUS 5.0 Programming software, Media Cybernetics, Inc). The percentages of positive nuclear staining (brown nuclei) were counted on 10 randomly selected fields in each compartment (seminiferous tubules and interstitial area) (Manee-in and Srisuwatanasagul, 2011). The results were presented as mean ratio of positive percentage nuclear staining. Statistical analysis: Data were handled and statistically analyzed using the GraphPad Prism (version 4, San Diego, USA). Normal distribution of residuals from the statistical models was tested using Kolmogorov-Smirnov test. Differences in mean number of positive percentage of ERβ, AR and Ki-67 were tested using Paired-T test. Correlation between positive percentage of ERβ, AR and Ki-67 were evaluated using Pearson correlation coefficients. A pvalue < 0.05 was considered statistically significant.
Results Microscopic evaluation: The similar histological structures of both scrotal and abdominal testicular tissues were observed. Both of testicular tissues were presented by small seminiferous tubules, which composed of spermatogonia and Sertoli cells. In addition, the clusters of Leydig cell were found in the interstitial area (Fig. 1). Pathological findings of both scrotal and abdominal testes were not observed.
Table 1 The percentage of Ki-67, ERβ and AR positive area (mean+SD) in the scrotal and abdominal testis of boar (n=8) Ki-67 ERβ AR Seminiferous Interstitial Seminiferous Interstitial area Seminiferous tubules area tubules tubules Scrotal testis 11.9±6.92 8.18±2.93 3.16±1.96 7.46±1.36a 4.96±1.96 Abdominal testis 9.83±2.01 7.56±1.83 3.71±3.12 2.97±1.89b 6.77±1.36 Overall significance p=0.40 NS NS p<0.05 NS Mean (+ SD) within the same column followed by the different superscript letters are significantly different (p<0.05) Tissues
Interstitial area 3.09±2.16 4.26±1.93 NS
Manee-in S. et al. / Thai J Vet Med. 2011. 41(1): 65-69.
Table 2 The percentage of Ki-67, ERβ and AR positive area (mean+SD) in the seminiferous tubules and interstitial space (n=8)
Area Seminiferous tubules Interstitial area Overall significance
Scrotal testis 11.9±6.92 8.18±2.93 NS
Ki-67 Abdominal testis 9.83±2.01 a 7.56±1.83 b p<0.05
ERβ Scrotal testis 3.16±1.96 7.46±1.36 NS
AR Abdominal testis 3.71±3.12 2.97±1.89 NS
Scrotal testis 4.96±1.96 3.09±2.16 NS
Abdominal testis 6.77±1.36 a 4.26±1.93 b p<0.01
Mean (+SD) within the same column followed by the different superscript letters are significantly different (p<0.05)
Immunohistochemistry: Immunolocalization of Ki-67 was observed in the nuclei of germ cells, interstitial cells, Sertoli cells and peritubular myoid cells of both scrotal and abdominal testicular tissues. For ERβ and AR, the immunolocalization was observed in the nuclei of germ cells and interstitial cells of both scrotal and abdominal testicular tissues (Fig. 2). The percentages of ERβ, AR and Ki-67 positive cells in scrotal and abdominal testes and the different compartments of testicular tissues are shown in table 1 and 2, respectively. In the interstitial area, a significantly higher positive percentage of ERβ was observed in the scrotal testis compared with the abdominal testis (p<0.05). In the scrotal testis, the percentage of ERβ positive in the interstitial cells appeared to be higher than in the seminiferous tubules (p=0.07).
Figure 1 The morphology of the abdominal and scrotal testicular tissue of prepubertal boar testis (A and B, respectively). Sertoli cells (arrow head), spermatogonia (black arrow) and Leydig cells (white arrow). Hematoxylin-eosin staining, Bar=100 µm
In the abdominal testis, a significantly higher number of AR positive cells were observed in the seminiferous tubules compared with the interstitial cells. In the scrotal testis, the percentage of AR positive cells of seminiferous tubules appeared to be higher than in the interstitial area (p=0.06). The percentage of Ki-67 positive cells in the seminiferous tubules was significantly higher than in the interstitial cells of abdominal testes (p=0.04). In the scrotal testis, the Ki-67 positive cells in the seminiferous tubules appeared to be higher than in the interstitial cells (p<0.1). A significant difference between percentage of Ki-67 positive cells in the scrotal and abdominal testes was not found. Therefore, there was no significant correlation between the positive area of ERβ, AR and Ki-67.
Discussion In the present study, similar histological findings were observed in both abdominal and scrotal testicular tissues. Both seminiferous tubules of the abdominal and scrotal testes were composed of immature Sertoli cells, a few spermatogonia and gonocyte. The Leydig cells (interstitial cells) were found in the interstitial area of the testicular tissues. These data suggested that the scrotal testicular tissues structure of prepubertal boar had not yet been fully developed (Ramesh et al., 2007). The expression of ERβ was observed in the nuclei of germ cells and interstitial cells. This finding is similar to the previous study of the testicular tissues of immature boar (Rago et al., 2004). In this study, the ERβ expression in the interstitial area of scrotal testis was significantly higher than in the abdominal testis. This finding indicated that scrotal testis might have a
Figure 2 The expression of ERβ, AR and Ki-67 protein in prepubertal boar testicular tissues. Positive immunostaining of ERβ, AR and Ki-67 in the seminiferous tubules (arrow head) and interstitial cells (black arrow) (A, C and E, respectively). Positive control sections for ERβ, AR and Ki-67 protein (black arrow) (B, D and F, respectively), Bar=100 µm
good opportunity to develop, since estrogens could modulate spermatogenesis and testicular development (Rago et al., 2004). The expression of ERβ was also observed in the germ cells, which can explain the role of estrogens in germ cells development (Carani et al., 1997). The expressions of AR in both abdominal and scrotal testes were not significantly different. This may be the result of the immaturity of the boars, in which the level of testosterone, which influence on the expression of AR, may be low (Ramesh et al., 2007). The expression of AR was pronounced in the
Manee-in S. et al./ Thai J Vet Med. 2011. 41(1): 65-69. seminiferous tubules than another area. Similar result was found in rat testicular tissue which indicated the role of androgen in spermatogenesis (Hansson et al., 1975). Ki-67 positive cells could be found in the seminiferous tubules and interstitial area of both the abdominal and scrotal testicular tissue. This is in accordance with previous study in cryptorchidism boar (Bernal-Manas et al., 2005). The Ki-67 positive cells were higher in the seminiferous tubules of the scrotal testis than in the abdominal testis, but there was not any significantly statistical difference. This finding may be explained by the similar histological structure of abdominal and scrotal testes found in the present study. However, when compared with an earlier study in cryptorchidism boar, the present study’s result is different. The earlier study showed a lower proliferation in abdominal testicular tissues (Bernal-Manas et al., 2005). The difference may be explained by the different structures between the abdominal and scrotal testis of postpubertal boars, in which scrotal testes are fully developed. Regarding testicular tissue compartments, the Ki-67 positive cells in the seminiferous tubules were higher than in the interstitial area. This finding suggested the role of germ cell proliferation that had a higher degree than the interstitial cells. In conclusion, this is the first report the steroid receptors protein expression in cryptorchidism testicular tissues of prepubertal boar. The expression of ERβ in the interstitial area was predominant in the scrotal testicular tissues than in the abdominal testicular tissue. A tendency of more expressions of Ki-67 receptors in the seminiferous tubule of the scrotal testicular tissues than in the abdominal testicular tissues was observed. However, the expression of AR protein in the scrotal and abdominal testicular tissues was not significant different. Therefore, a further study of steroid hormone receptors, especially AR, in cryptorchidism boar should be conducted in older boars which have fullydeveloped scrotal testicular structures for a better understanding of the pathogenesis and physiological status of cryptorchidism boar.
Acknowledgement This study was supported by a grant from Faculty of Veterinary Science, Mahidol University. We thank Dr. Walasinee Moonarmart for statistical assistance.
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69 372. Carani, C., Qin, K., Simoni, M., Faustini-Fustini, M., Serpente, S., Boyd, J., Korach, K.S. and Simpson, E.R. 1997. Effect of testosterone and estradiol in a man with aromatase deficiency. N Engl J Med. 337(2): 91-95. Gerdes, J., Lemke, H., Baisch, H., Wacker, H.H., Schwab, U. and Stein, H. 1984. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 133(4): 1710-1715. Gerdes, J., Li, L., Schlueter, C., Duchrow, M., Wohlenberg, C., Gerlach, C., Stahmer, I., Kloth, S., Brandt, E. and Flad, H.D. 1991. Immunobiochemical and molecular biologic characterization of the cell proliferationassociated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 138(4): 867-873. Hansson. V., Weddington. S.C., McLean. W.S., Smith, A.A., Nayfeh, S.N., French, F.S. and Ritzen, E.M. 1975. Regulation of seminiferous tubular function by FSH and androgen. J Reprod Fertil. 44(2): 363-375. Manee-in, S. and Srisuwatanasagul, S. 2011. Proliferation and apoptosis in normal bitch mammary tissues in relation to progesterone level. Comp Clin Path. DOI: 10.1007/s00580010-1144-9 Pinart, E., Bonet, S., Briz, M., Pastor, L.M., Sancho, S., Garcia, N., Badia, E. and Bassols, J. 2002. Histochemical study of the interstitial tissue in scrotal and abdominal boar testes. Vet J. 163(1): 68-76. Pinart, E., Sancho, S., Briz, M.D., Bonet, S. and Badia, E. 1999a. Efficiency of the process of meiosis in scrotal testes of healthy boars and unilateral abdominal cryptorchid boars. Teratology. 60(4): 209-214. Pinart, E., Sancho, S., Briz, M.D., Bonet, S. and Garcia, N. 1999b. Characterization of the semen quality of postpuberal boars with spontaneous unilateral abdominal cryptorchidism on the right side. Anim Reprod Sci. 55(3-4): 269-278. Rago, V., Maggiolini, M., Vivacqua, A., Palma, A. and Carpino, A. 2004. Differential expression of estrogen receptors (ERalpha/ERbeta) in testis of mature and immature pigs. Anat Rec A Discov Mol Cell Evol Biol. 281(2): 1234-1239. Ramesh, R., Pearl, C.A., At-Taras, E., Roser, J.F. and Berger, T. 2007. Ontogeny of androgen and estrogen receptor expression in porcine testis: effect of reducing testicular estrogen synthesis. Anim Reprod Sci. 102(3-4): 286-299. Sriraman, V., Rao, V.S., Sairam, M.R. and Rao, A.J. 2000. Effect of deprival of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and IGF-1. Mol Cell Endocrinol. 162(1-2): 113120. Wang, R.S., Yeh, S., Tzeng, C.R. and Chang, C. 2009. Androgen receptor roles in spermatogenesis and fertility: lessons from testicular cell-specific androgen receptor knockout mice. Endocr Rev. 30(2): 119-132.
Improvement of Normal Fertilization Rate and Embryo Development by Reduction of Sperm: Oocyte ratio during In vitro Fertilization in Pig Sasithorn Panasophonkul1, 2 Theerawat Tharasanit1 Panida Chanapiwat1 Mongkol Techakumphu1*
Abstract The present study was undertaken to determine the influence of sperm:oocyte ratio at fertilization in vitro using frozen boar semen on fertilization rates and subsequent embryo development and quality in pig. Cumulusoocyte complexes (COCs) were collected from porcine ovaries and then matured in vitro. After 44 hours of culture, matured oocytes were fertilized for 6 hours with three different sperm:oocyte ratios (1000:1, 2000:1, and 4000:1). Presumptive zygotes were fixed at 18-20 hours post-fertilization and then examined for sperm penetration and monospermy rates. The developmental competence, in terms of cleavage and blastocyst rates, and the blastocyst quality were evaluated at 48 and 168 hours post-fertilization, respectively. Sperm penetration rate significantly increased when the oocytes were fertilized with 2000 (90.23±2.5%) and 4000 (93.46±3.7%) sperm: oocyte, compared with those fertilized with 1000 (74.08±1.2%) sperm:oocyte (p=0.005). The oocytes inseminated with 1000 sperm per oocyte had a significantly higher rate of monospermic zygotes (81.79±2.9%) than those inseminated with 2,000 and 4,000 sperm per oocyte (48.07±6.0 and 31.51±4.9%, respectively; p=0.001). The development of blastocysts increased significantly (p<0.05) in the group fertilized with 1,000 sperm:oocyte (29.02±1.8%) compared to those of 4,000 sperm:oocyte (14.00±3.0%). However, there was no significant difference in the mean number of blastocyst cells among the groups but blastocysts derived from 1000:1 oocyte/sperm ratio had the highest ICM cell numbers. Our results indicated that optimization of sperm:oocyte ratio at fertilization in vitro improved fertilization rates, in particular, the monospermic penetration rate and blastocyst rate. Keywords: in vitro fertilization, pig, sperm:oocyte ratio 1Department of Obstetrics, Gynaecology and Reproduction, Chulalongkorn University, Faculty of Veterinary Science, Bangkok 10330, Thailand 2Equine Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 71-77.
Panasophonkul S. et al. / Thai J Vet Med. 2011. 41(1): 71-77.
บทคัดย่อ การเพิ่มของอัตราการปฏิสนธิและการพัฒนาของตัวอ่อนโดยการลดอัตราส่วนจํานวนตัวอสุจิต่อ โอโอไซต์ในระหว่างการปฏิสนธิภายนอกร่างกายในสุกร ศศิธร พนโสภณกุล 1, 2 ธีรวัฒน์ ธาราศานิต 1 พนิดา ชนาภิวัฒน์ 1 มงคล เตชะกําพุ 1* การศึกษาครั้งนี้มีจุดประสงค์เพื่อศึกษาผลของอัตราส่วนจํานวนตัวอสุจิต่อโอโอไซต์ในช่วงการปฏิสนธิภายนอกร่างกายต่ออัตราการ ปฏิสนธิ ความสามารถในการพัฒนาและคุณภาพของตัวอ่อนที่ได้เก็บโอโอไซต์ที่ถูกหุ้มล้อมด้วยกลุ่มเซลล์คูมูลัสจากรังไข่สุกร และเลี้ยงใน น้ํายาเลี้ยงนาน 44 ชั่วโมง แบ่งโอโอไซต์ที่มีการเจริญเต็มที่ออกเป็น 3 กลุ่มตามอัตราส่วนจํานวนตัวอสุจิต่อโอโอไซต์ที่ใช้ในการปฏิสนธิ คือ 1000:1 2000:1 และ 4000:1 นาน 6 ชั่วโมง และทําการเลี้ยงในน้ํายาเลี้ยงตัวอ่อน ตรึงตัวอ่อนบางส่วนที่ 18 ชั่วโมงหลังการปฏิสนธิ เพื่อ ประเมินอัตราการผ่านเข้าปฏิสนธิของตัวอสุจิ และการเข้าปฏิสนธิของตัวอสุจิหนึ่งตัวต่อโอโอไซต์ (Monospermic oocyte) จากนั้นประเมิน ความสามารถในการแบ่งตัวและเจริญเป็นระยะคลีเวทและระยะบลาสโตซิสหลังการปฏิสนธิที่ 48 และ 168 ชั่วโมง ตามลําดับ และคุณภาพ ของตัวอ่อนที่ผลิตได้ ผลการศึกษาพบว่าโอโอไซต์ที่ทําการปฏิสนธิด้วยจํานวนตัวอสุจิ 2000 และ 4000 ตัวต่อโอโอไซต์ มีอัตราการผ่านเข้า ปฏิสนธิของตัวอสุจิมากกว่าโอโอไซต์ที่ทําการปฏิสนธิด้วยจํานวนตัวอสุจิ 1000 ตัวต่อโอโอไซต์อย่างมีนัยสําคัญ(p=0.005) และกลุ่มโอโอไซต์ ที่ได้รับการปฏิสนธิด้วยจํานวนตัวอสุจิ 1000 ตัวต่อโอโอไซต์มีอัตราการเข้าปฏิสนธิของตัวอสุจิหนึ่งตัวต่อโอโอไซต์สูงกว่ากลุ่มการทดลองอื่น อย่างมีนัยสําคัญ (p=0.001) นอกจากนี้ยังพบว่ามีอัตราการเจริญของตัวอ่อนระยะบลาสโตซิสสูงกว่ากลุ่มของโอโอไซต์ที่ทําการปฏิสนธิด้วย จํานวนตัวอสุจิ 4000 ตัวต่อโอโอไซต์ (p<0.05) แต่ไม่พบว่ามีความแตกต่างของค่าเฉลี่ยจํานวนเซลล์ของตัวอ่อนระยะบลาสโตซิสในแต่ละ กลุ่มทดลอง จากผลการศึกษาสรุปได้ว่าอัตราส่วนของจํานวนตัวอสุจิต่อโอโอไซต์ที่เหมาะสมในช่วงการปฏิสนธิภายนอกร่างกายสามารถเพิ่ม อัตราการปฏิสนธิโดยเฉพาะอย่างยิ่งอัตราการเข้าปฏิสนธิของตัวอสุจิหนึ่งตัวต่อโอโอไซต์และอัตราการเจริญของตัวอ่อนระยะบลาสโตซิส คําสําคัญ: การปฏิสนธิภายนอกร่างกาย สุกร อัตราส่วนจํานวนตัวอสุจติ ่อโอโอไซต์ 1 ภาควิชาสูติศาสตร์ เธนุเวชวิทยาและวิทยาการสืบพันธุ์ คณะสัตวแพทยศาสตร์ จุฬาลงกรณ์มหาวิทยาลัย กรุงเทพฯ 10330 2 คลินิกม้า คณะสัตวแพทยศาสตร์ มหาวิทยาลัยเชียงใหม่ เชียงใหม่ 50100 *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction In vitro embryo production (IVP) techniques consisting of oocyte maturation, fertilization and embryo culture have been used to generate a large quantity of embryos for fundamental and applied biomedical research such as studying the developmental biology of embryos and also xenotransplantation. Until recently, embryonic stem (ES) cell, derived from porcine blastocyst, hold a tremendous interest for cell replacement therapy because it has been shown to be a better model for human diseases compared to other small laboratory animals (Ibrahim et al., 2006; Matsunari and Nagashima, 2009). It is therefore not surprising that researchers over the world have increasingly attempted to generate porcine embryonic stem cells although overall success has been markedly restricted to only porcine embryonic-like stem cells (Li et al., 2004; Brevini et al., 2010). In vitro fertilization (IVF) technique has been considered as a suitable technique in producing
blastocyts for ES cell establishment when compared with parthenogenetic activation (PA) technique (Panasophonkul et al., 2010). This non-invasive and cost-effective technique provides viable embryos similar to its in vivo-derived counterparts and also has advantages over in vivo embryos by the large-scale embryo production with less time consuming. However, multiple sperm penetration (polyspermy) is still a major problem of IVF in pig (McCauley et al., 2003; Kikuchi et al., 2004; Sherrer et al., 2004; Koo et al., 2005). Previous studies have demonstrated a high correlation between the frequency of polyspermy and the absolute number of inseminated spermatozoa (i.e. sperm:oocyte ratio), rather than the number of spermatozoa per milliliter of IVF medium (Xu et al., 1996; Rath, 1992). IVF with excessive sperm numbers have elevated the frequency of polyspermic penetration (Abeydeera and Day, 1997; Marchal et al., 2002; Matas et al., 2003) that coincides with a low rate of embryo development in vitro, especially at the blastocyst stage (Machaty et al., 1998; Han et al., 1999).
Panasophonkul S. et al./ Thai J Vet Med. 2011. 41(1): 71-77. Furthermore, this polyspermic penetration also decreases embryo quality in terms of total cell numbers and inner cell mass (ICM) ratio when compared with monospermic blastocysts (Giles and Foote, 1995; Funahashi and Day, 1997). Boars and ejaculate variation appeared to influence the incidence of penetration, fertilization and embryo quality when fresh semen is used (Xu et al., 1996; Long et al., 1999). Suzuki et al., (2003) revealed that penetration and polyspermy rates were more variable among the breeds than among boars within a breed. In contrast to fresh semen, the use of frozen-thawed sperm from the same boar eliminates the variation between trials, providing much more reproducible data and the possibility of repeating the experiment (Funahashi and Nagai, 2001; Marchal et al., 2002; Gil et al., 2004). The present study was undertaken to determine the influence of sperm:oocyte ratio at fertilization in vitro on fertilization rate and subsequent embryo development and quality in order to produce a high number and quality of porcine blastocysts.
Materials and Methods All chemicals used in this study were purchased from the Sigma-Aldrich (St. Louis, USA) unless otherwise stated. Collection and culture of oocytes: The collection and in vitro maturation (IVM) of oocytes were performed as previous described (Panasophonkul et al., 2010). Briefly, cumulus oocyte complexes (COCs) were aspirated from antral follicles (∅ 3-8 mm). COCs with homogeneous cytoplasm surrounded with at least three uniform layers of compact cumulus cells were selected and cultured for in vitro maturation, groups of 30-50 COCs were cultured at 38.5°C in a humidified atmosphere with 5% CO2 in air for 44 hours in a maturation medium that consisted of TCM199 with Earle’s salts, 3.05 mM glucose, 26.2 mM sodium bicarbonate, 0.69 mM L-glutamine, 0.91 mM sodium pyruvate, 0.1 mM cysteamine, 10 ng/ml epidermal growth factor (EGF), 100 IU/ml penicillin, 100 µg/ml streptomycin and 10% (v/v) porcine follicular fluid (pFF).The IVM medium was supplemented with 10 IU/ml equine chorionic gonadotropin (Folligon®, eCG, Intervet-Schering-Plough Animal Health, Boxmeer, The Netherlands) and 10 IU/ml human chorionic gonadotropin (Chorulon®, hCG, IntervetSchering-Plough Animal Health) for the first 22 hours. The oocytes were then additionally cultured for a further 22 hours in an absence of eCG and hCG. In vitro fertilization: After 44 hours of in vitro maturation, expanded cumulus cells were partially removed and then washed three times with preequilibrated modified Tris-buffered medium (mTBM) (Abeydeera and Day, 1997) supplemented with 5 mM sodium pyruvate, 100 IU/ml penicillin and 100 µg/ml streptomycin (IVF medium). Groups of 30-50 oocytes were placed into a 4-well plate containing 500 μl of IVF medium and incubated for at least 30 hours before fertilization. Oocytes were coincubated with frozen-thawed spermatozoa for 6 hours at 38.5oC in a
73 humidified atmosphere with 5% CO2 in air. Sperm preparation: The frozen semen from a single boar (Yorkshire) was thawed for 12 sec at 50oC. The viable and motile sperm were then selected using the Percoll gradient technique as described by Parrish et al. (1994). Briefly, the contents of semen were layered onto a discontinuous gradient of 45% (v/v) and 90% (v/v) Percoll in a 15 ml-conical tube (BD FalconTM, Spark, MD, USA) and then centrifuged at 26oC, 700 x g for 15 min. The sperm pellet at the bottom of the 90% Percoll fraction was slowly resuspended with 1 ml of IVF medium. After re-centrifugation, the sperm pellet was resuspended in IVF medium and the concentration of spermatozoa was calculated. The sperm motility was subjectively assayed at x100 magnification under a light microscope (TS1000 Nikon, Tokyo, Japan). Percoll-treated spermatozoa to be used for the experiment had more than a 90% progressive motility. Embryo culture: After IVF, oocytes surrounded with cumulus cells were denuded and washed three times in an in vitro culture (IVC) medium (North Carolina State University (NCSU)-23; Petters and Wells, 1993), supplemented with 1% (v/v) non-essential amino acids (NEAA) and 4 mg/ml bovine serum albumin (BSA). Groups of 30-50 denuded oocytes were placed into a 4-well plate (Nunc, NY, USA) containing 500 μl of NCSU-23 and then covered with mineral oil. During the 5th to 7th days of embryo culture, the BSA in NCSU-23 was substituted with 10% (v/v) heatinactivated fetal bovine serum (FBS; Gibco, Invitrogen USA). In all cases, embryos were cultured at 38.5ºC in a humidified atmosphere with 5% CO2 in air. Assessment of sperm penetration: Of 940 IVM oocytes inseminated with three different sperm: oocyte ratios, 243 oocytes were fixed and stained at 18-20 hours after culture to assess fertilization parameters. Presumptive zygotes were washed twice in phosphate buffered saline (PBS) supplemented with 0.1% (w/v) BSA and then were fixed in 4% (w/v) paraformaldehyde at room temperature for 15 min. To assess the nuclear status of presumptive zygotes, they were first stained with 1 µg/ml fluorescent DNA labeling (4’, 6’ Diamidino-2phenylindole dihydrochloride: DAPI) for 15 min. The fluorescently labeled zygotes were mounted on a glass microscope slide in a 2 µl droplet of anti-fade medium (VectashieldTM, Vector Lab, CA, USA) to retard photobleaching, and then examined under an epifluorescent microscope (BX51 Olympus, Japan) at x 200 and x 400 magnifications . Sperm penetration of porcine oocytes was classified by the presence of at least one male pronucleus that had formed in the ooplasm. Monospermic fertilization typified by the presence of only one male pronucleus, while multiple pronuclei (more than 2) indicated polyspermic fertilization.
Differential staining of blastocysts: Differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells was performed as our
Panasophonkul S. et al. / Thai J Vet Med. 2011. 41(1): 71-77. Effect of sperm:oocyte parameters
previous description (Panasophonkul et al., 2010). Fluorescently labeled embryos were mounted onto a glass microscopic slide in a droplet of anti-fade medium (VectashieldTM, CA, USA) and sealed with a coverslip. The embryos were examined and counted for ICM and TE cell numbers using an epifluorescent microscope. The ICM was classified as a group of embryonic cells that was stained with only Hoechst 33342 (blue), while the TE cells were positive to both Hoechst 33342 and propidium iodide.
There was a significant effect of sperm:oocyte ratio on penetration rate, monospermy, and the efficiency of fertilization (Table 1). Our results showed that sperm penetration rate significantly increased when the oocytes were fertilized with 2000 (90.23±2.5%) and 4000 (93.46±3.7%) sperm: oocyte, compared with those fertilized with 1000 (74.08±1.2%) sperm:oocyte (p=0.005). However, the increase in sperm penetration rate was associated with a high rate of polyspermy (multiple pronucleus) since the oocytes inseminated with 1000 sperm per oocyte had a significantly higher rate of monospermic zygotes (81.79±2.9%) than those inseminated with 2,000 and 4,000 sperm per oocyte (48.07±6.0 and 31.51±4.9%, respectively; p=0.001, Fig. 1). The efficiency of fertilization also significantly increased when 1000 spermatozoa per oocyte were used, compared to those 4000 sperm per oocyte (60.64±2.7 versus 29.36±4.8%; p=0.002). However, the fertilization efficiency was not significantly different between 2000 and 4000 sperm:oocyte groups.
Experimental design: In order to examine the effect of sperm numbers on the percentage of normal fertilization (monospermic fertilization), the in vitro matured oocytes were fertilized with three different sperm:oocyte ratios (1000:1, 2000:1, and 4000:1). The frozen-thawed sperm used in this experiment were obtained from a single boar and the sperm quality following Percoll gradient treatment by means of progressive motility was presumed to be equal in all replicates. The presumptive embryos were either fixed at 18-20 hours post-IVF for examination of pronuclear formation or further cultured for 7 days in order to determine their developmental competence (cleavage and blastocyst formation rates) and embryo quality (ICM, TE and ICM: total cells ratio). Statistical analysis: Data expressed by mean ± standard error of the mean (SEM) was pooled from at least 4 independent replicates. Fertilization parameters, developmental competence and quality of IVF embryos among the experimental groups were compared by ANOVA and protected least significant different (LSD) statistical tests. Statistical analysis was carried out with SPSS version 13.0 software (SPSS Inc., Chicago, IL). P<0.05 was considered statistically significant.
Figure 1 Pronuclear formation of fertilized oocytes at 18-20 hours post-IVF. A) Monospermic fertilization typified by the presence of only one male (arrowhead) and one female pronucleus (arrow) while polyspermic fertilization; B) presents more than one of male pronucleus (arrowheads). (x200).
Table 1 Effect of sperm:oocyte ratios on fertilization parameters during IVF of pig oocytes matured in vitro Percentage of oocytes (mean+SEM) Ratio
No. of inseminated oocytes
% Efficiency** (mean+SEM)
*Percentage of the number of monospermic oocytes/total of penetrated oocytes; ** percentage of the number of monospermic oocytes/total of inseminated oocytes. a, b, c within a column, different superscripts denote values that differ significantly (p<0.05 at least).
Table 2 Effect of different sperm:oocyte ratios on the cell numbers of blastocysts No. of cells (mean+SEM)
No. of blastocysts
ICM : Total cell (%mean+SEM)
ICM: inner cell mass and TE: trophectoderm
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Figure 2 Effect of sperm:oocyte ratios on developmental competence of embryos at cleavage and blastocyst stages after IVF
Development and quality of IVF embryos after fertilization with different sperm:oocytes ratios Cleavage and blastocyst development was observed at 48 and 168 hour after IVF, respectively. As shown in Fig 2, the sperm:oocyte ratio during IVF did not affect the cleavage rate, while the blastocyst rate increased significantly (p<0.05) in the group fertilized with 1,000 sperm:oocyte (29.02±1.8%) compared to those 4,000 sperm:oocyte (14.00±3.0%). Moreover, we examined the quality of blastocysts in terms of the mean number of cells including ICM and TE. Although there was no statistical difference in the mean number of blastocyst cells (p>0.05) among the experimental groups, the blastocysts derived from oocytes inseminated with 1000 sperm:oocyte had a higher tendency of ICM cell numbers than thoseobtained from the higher sperm:oocyte ratios (Table 2).
Discussion In this study, we demonstrate that sperm: oocyte ratio at in vitro fertilization affects the efficiency and quality of embryos. A decresing sperm:oocyte reduced the percentage of polyspermy and also improved the developmental competence. This work is correlated with previous studies reporting that excessive numbers of spermatozoa at the time of fertilization increase the percentage of polyspermic penetration (Rath, 1992; Xu et al., 1996; Gil et al., 2007), thereby impairing embryo development. In the current study, frozen-thawed semen from a single donor was treated with Percoll gradient density in order to minimize boar-to-boar and sperm quality variations during the experiments. Percoll treatment has been reported to improve sperm viability and quality in bovines (Somfai et al., 2002; Mendes et al., 2003) and porcines (Jeon and Yang, 2001; Matas et al., 2003). When IVF was carried out with different sperm: oocyte ratios for 6 hours, our results showed that the rates of sperm penetration were significantly enhanced by increasing the ratios of sperm number from 2000 to 4000 per oocyte, compared with the 1000 sperms per oocyte group. Although we were not able to decrease the number of
75 inseminated sperm to lower than 1000 sperm per oocytes, our results showed that the monospermic penetration rate (81.79%) was significantly high when partial cumulus-enclosed oocytes were co-incubated with 1000 sperm per oocyte. These results were similar to previous reports indicating that a relative reduction of spermatozoa numbers during in vitro fertilization results in greater monospermic penetration rates (Xu et al., 1996; Abeydeera and Day, 1997; Gil et al., 2004). Rath (1992) revealed a high correlation between polyspermy and the absolute number of spermatozoa and oocytes but not between the polyspermy rate and sperm concentration per milliliter. Our results obtained a higher incidence of monospermy (82%) when 1000 sperm per oocyte were used for IVF compared with 50-60% from other studies. The observed differences might be caused by many factors such as sperm quality (Popwell and Flowers, 2004; Gadea, 2005) and the inter- and intraboar variability (Suzuki et al., 2003). In addition, suboptimal oocyte maturation and IVF systems caused by culture medium and/or sperm incubation time have also been demonstrated to involve polyspermic penetration (reviewed by Funahashi, 2003). Until recently, the mechanism for the prevention of multiple sperm entry has not been well understood. It has been demonstrated that normal distribution of intracellular organelles of mitochondria and cortical granules (CG) during IVM played an important role in preventing polyspermy (Cran and Cheng, 1986; Grupen et al., 1997). More specifically, the CG contents released into the perivitelline space (PVS) of the fertilized oocyte induce hardening of the zona pellucida (ZP), thereby preventing polyspermy. In this respect, a delay and incomplete exocytosis of CG due principally to poor cytoplasmic maturation causes an improper pattern of cortical distribution and exocytosis (Wang et al., 1997). In addition, the forming of narrowed PVS in in vitro matured oocytes caused by culture medium may also interfere with the distribution of CG contents and could also delay the zona block (Funahashi et al., 1994). In the present study, we designed to use partial cumulus-enclosed oocytes instead of cumulusdenuded oocytes because the presence of cumulus cells surrounding the oocyte during the IVF has been demonstrated to be beneficial to a normal fertilization rate in several species (Vanderhyden and Armstrong, 1989; Kikuchi et al., 1993; Wang and Niwa, 1995; Zhang et al., 1995) principally by secreting the substances that promote penetration and acrosome reaction of sperm. Cumulus cells also provide a bidirectional microenvironment for attracting, trapping and the selection of morphologically normal and hyperactive spermatozoa during IVF (Lavy et al., 1988; Tanghe et al., 2002). In this study, we did not found any difference in the cleavage rates among the three different sperm:oocyte ratios. However, the number of cleaved embryos that developed to blastocyst stage was significantly higher in oocytes fertilized with the lowest sperm:oocyte (1000:1) ratio than those
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fertilized with greater sperm:oocyte (4000:1) ratio (29 and 14%, respectively). These observations were similar to a study demonstrating that fewer than 20% of inseminated oocytes developed to blastocyst stage when a high sperm concentration was used (Koo et al., 2005). However, there was no significantly difference in embryo quality among experimental groups although the blastocysts obtained from low sperm number insemination tended slightly to increase the ICM cell numbers which was different from a report indicating the retard development of the ICM in polyploidy embryos (Han et al., 1999). This indicates that other factors may also influence ICM segregation and self-renewal. It is also likely that embryos have intrinsic ability to control the optimal number of ICM probably via programmed cell death (Hardy et al., 2003) and gene regulating pathways (Marikawa and Alarcón, 2009). In conclusion, our findings demonstrate that optimization of sperm: oocyte ratio during in vitro fertilization improves fertilization rates and, in particular, monospermic penetration and the quality of blastocysts. Nevertheless, other factors involved in the IVM-IVF system should be considered to increase the success rates of porcine IVF.
Acknowledgement This work was financially supported by Commission on Higher Education, Chulalongkorn University Centenary Academic Development Project, The National Research University Project of CHE and The Ratchadaphiseksomphot Endownment Fund (HR1116I), PS is PhD student in CHE-PhD program, Thailand.
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Abstract Mesenchymal stem cells (MSCs) are multipotent cells that have characteristics of self-renewal and differentiation into various specific cell types, in particular mesodermal lineages. This study aimed at isolating, identifying and examining the differentiation capability of canine MSCs. Bone marrow aspirates were obtained from 4 dogs. Putative MSCs were then cultured in MSC medium and subpassaged. At the 3rd to 5th passages, MSCs were examined for their morphology and doubling time. Two cell lines were examined for the expression of CD 34, CD 44 and CD 90, using flow cytometry. The in vitro differentiation of these MSCs into mesodermal lineages (bone, cartilage and adipose tissues) and ectodermal lineage (neuron) was performed using osteogenic, chondrogenic, adipogenic and neurogenic media, respectively. Histological examinations (Von Kossa and alcian blue staining) and mRNA expressions (GLA and COL1A1) were used to examine the bone and cartilage differentiation, while Oil red O staining was used to determine adipogenic differentiation. Plastic-adhered MSCs had high potential for cell division, with a mean doubling time of 35.4±9.3 hours. These fibroblast-like MSCs expressed MSCs markers (CD 44 and CD 90), while fewer than 5% of these MSCs were tested positive to a hemopoietic stem cell marker (CD34). Based on the histological examinations and gene expressions, these cells demonstrated the ability to differentiate into bone, cartilage and adipose tissues. In conclusion, MSCs can be isolated from canine bone marrow and these cells are capable of in vitro differentiation into specific mesodermal lineages. Keywords: bone marrow, canine, mesenchymal stem cells 1Department
of Obstetrics, Gynaecology and Reproduction, 2 Department of Veterinary Surgery, 3Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand 4The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 79-86.
Tharasanit T. et al. / Thai J Vet Med. 2011. 41(1): 79-86.
บทคัดย่อ ประสิทธิภาพการเจริญเปลี่ยนแปลงของเซลล์ต้นกําเนิดมีเซนไคม์จากสุนขั ธีรวัฒน์ ธาราศานิต 1* นวเพ็ญ ภูติกนิษฐ์ 1 ชาลิกา หวังดี 2 กัมปนาท สุนทรวิภาต 2 ศศิจรัส ตันตระจักร 2 ธีระยุทธ แก้วอมตะวงค์ 3 จันเพ็ญ สุวิมลธีระบุตร 1 พิชญ์ ศุภผล 4 มงคล เตชะกําพุ 1* เซลล์ต้นกําเนิดมีเซนไคม์ (MSCs) เป็นเซลล์ที่มีความสามารถในการเจริญเพิ่มจํานวนได้เองและสามารถพัฒนาเป็นเซลล์จําเพาะ หลายชนิดโดยเฉพาะเซลล์ในกลุ่มเนื้อเยื่อชั้นกลาง การศึกษาครั้งนี้มีวัตถุประสงค์ เพื่อศึกษาวิธีการเก็บ การพิสูจน์ และการตรวจสอบ คุณสมบัติการเจริญเปลี่ยนแปลงของเซลล์ต้นกําเนิดมีเซนไคม์ของสุนัข ภายหลังการเก็บของเหลวจากโพรงกระดูกสุนัข ทําการเลี้ยงเซลล์เพื่อ เพิ่มจํานวนและทําการตรวจลักษณะรูปร่างและอัตราเร็วของการเพิ่มจํานวนเซลล์เป็นสองเท่า จากนั้นทําการตรวจการแสดงออกของโปรตีน บนผิวเซลล์ชนิด CD 34 CD 44 CD 90 ด้วยเทคนิคโฟลวไซโตเมตรี และตรวจคุณสมบัติของเซลล์ในการเจริญเปลี่ยนแปลงเป็นเซลล์ในกลุ่ม เนื้อเยื่อชั้นกลาง (กระดูก กระดูกอ่อน และ ไขมัน) และเนื้อเยื่อชั้นนอก (เซลล์ประสาท) ใช้เทคนิคทางจุลพยาธิวิทยาร่วมกับการย้อมสี Von Kossa และ Alcian blue รวมถึงการแสดงออกของ mRNA ต่อยีน GLA และ COL1A1 ในการตรวจวินิจฉัยกระดูกและกระดูกอ่อน ตามลําดับ และใช้ Oil Red O ในการตรวจเนื้อเยื่อไขมัน เซลล์ MSCs ที่เกาะบนจานเพาะเลี้ยงพลาสติกมีคุณสมบัติในการแบ่งตัวโดยมีค่าเฉลี่ยของระยะเวลาการเพิ่มจํานวนเป็นสองเท่า เท่ากับ 35.4±9.3 ชั่วโมง เซลล์ MSCs มีลักษณะคล้ายเซลล์ไฟโบรบลาส ให้ผลบวกต่อการแสดงออกของโปรตีนจําเพาะต่อเซลล์ MSC (CD 44 และ CD 90) ในขณะที่เซลล์จํานวนน้อยกว่าร้อยละ 5 ให้ผลบวกต่อเซลล์ต้นกําเนิดเลือด (CD 34) จากการตรวจคุณสมบัติของเซลล์ด้วย เทคนิคทางจุลพยาธิวิทยาและการแสดงออกของยีน พบว่าเซลล์เหล่านี้สามารถเจริญพัฒนาเป็นเซลล์กระดูก กระดูกอ่อน และ เซลล์ไขมันได้ การศึกษาครั้งนี้สรุปว่าเซลล์ MSCs ที่เจาะเก็บจากโพรงกระดูกสุนัขมีความสามารถในการเจริญพัฒนาในจานเพาะเลี้ยงให้เป็นเซลล์ใน กลุ่มเนื่อเยื่อชั้นกลางได้ คําสําคัญ: ไขกระดูก สุนัข เซลล์ต้นกําเนิดมีเซนไคม์ 1 ภาควิชาสูติศาสตร์ เธนุเวชวิทยาและวิทยาการสืบพันธุ์ 2 ภาควิชาศัลยศาสตร์ 3ภาควิชาพยาธิวิทยา คณะสัตวแพทยศาสตร์ 4 วิทยาลัยปิโตรเลียมและปิโตรเคมี จุฬาลงกรณ์มหาวิทยาลัย กรุงเทพฯ 10330 *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction Stem cells have been intensively studied over the past two decades because these cells have a remarkable potential to develop into various specific cell lineages upon being cultured in appropriated conditions. Because of this, they have been considered as a powerful tool for cell- or tissue-based engineering in human and veterinary medicine (Barry et al., 2004; Bongso et al., 2008; Ribitsch et al., 2010). Stem cells are different from other cell types by two important characteristics (Wobus and Boheler, 2005), as they have the capability of self-renewal, while maintaining themselves in an undifferentiated stage. In specific condition, these quiescent stem cells, however, differentiate into specific cell types or tissues with special functions such as cardiomyocyte (Jing et al., 2008; Mayorga et al., 2009), bone (Barry and Murphy, 2004), cartilage (Kavalkovich et al., 2002) and neuron (Trzaska et al., 2007; Kim et al., 2009).
Stem cells are usually classified into three types (viz. embryonic, induced-pluripotent and adult stem cells), according to their origins and production techniques. Embryonic stem (ES) cells are pluripotentspecialized cells that are isolated from an inner cell mass of blastocyst-stage embryos (Evans and Kaufman, 1981; Martin, 1981). Induced-pluripotent stem cells (iPS) are also pluripotent stem cells that can be derived from genetic modification of nonpluripotent somatic cells (Takahashi and Yamanaka, 2006). Although these two types of ES cells are capable of unlimited cell division and differentiation into all three germ layers (endoderm, ectoderm and mesoderm), their clinical exploitation has been obscured by the possibility of tumorogenesis after transplantation in vivo (Arnhold et al., 2000; Reubinoff et al., 2000; Erdo et al., 2003). In contrast to the ES cells, adult stem cells have less ability of self-renewal, and their differentiation usually occurs within a cell lineage from which they originated. For example, mesenchymal stem cells (MSCs) can only differentiate
Tharasanit T. et al. / Thai J Vet Med. 2011. 41(1): 79-86. into mesodermal lineages, such as bone, cartilage and adipose tissues (Zuk et al., 2002). Interestingly, although these cells have been described as multipotent stem cells as differentiation potentials are essentially restricted to only mesodermal lineages, a recent report showed that they also have a potential to differentiate into other cell lineages, such as endoderm and ecdoderm origins (a process referred to as transdifferentiation) (Alaminos et al., 2010). To date, host-specific MSCs are highly desired in regenerative medicine because they can be logically isolated and propagated from many tissue origins, such as bone marrow and adipose tissue. Canine MSCs have been demonstrated to have the potential for use in cell-based therapy, particularly for bone and soft tissue regeneration (Kraus and KirkerHeadm, 2006; Hiyama et al., 2008; Jang et al., 2008; Jung et al., 2009; Zucconi et al., 2010). It is commonly accepted that the identification of MSCs relies on the expressions of positive (Stro-1, CD 90, CD 105, CD 44, CD 73) and negative markers (i.e., markers for hemopoietic cells: CD 34 and 45). In dogs, only the attachment property of MSCs to plastic culture dishes is commonly-accepted method for the selection of canine MSCs, while the use of MSC markers for identification varies from one laboratory to the next. The present study was aimed at evaluating the isolation and the identification techniques for canine mesenchymal stem cells (MSCs) that are derived from bone marrow aspirates and studying their differentiation potentials.
Materials and Methods All chemicals were purchased from SigmaAldrich, St Louis, USA, unless otherwise specified. Isolation of mesenchymal stem cells from bone marrow aspirates: Bone marrow aspirates were obtained from 4 healthy dogs. The procedure for obtaining these aspirates was reviewed and approved by the Ethical Committee for Animal Use, Faculty of Veterinary Science, Chulalongkorn University (Accession No. 0931055). In brief, the animals were premedicated intramuscularly with 0.1 mg/ml Acepromacine maleate (VetranquilTM; Ceva Sante animal, Libourne, France) and 0.25 mg/kg morphine sulphate (Food and Drug Administration, Bangkok, Thailand). After 15-20 min, anesthesia was induced intravenously with 4 mg/kg propofol (Fresenius Kabi Austria GmbH, Graz, Austria). The bone marrow contents were collected from either the humorous or iliac crest into a 10-ml heparinized syringe (containing 1000 IU heparin). The bone marrow aspirates were then transported to the laboratory (at 26°C) and processed within 4 hours after bone marrow aspiration. Upon arrival, the bone marrow aspirates were first layered onto gradient density (Histopaque®-1077 density 1.077 g/ml) and centrifuged at 26°C and 400g for 30 min. The mononuclear cells at the interface between each of the bone marrow aspirates and Histopaque® were used. Occasionally, remaining red blood cells were mixed and incubated with an equal volume of red blood cell lysis buffer for 5 min. The mixture was then
81 centrifuged and resuspended with 1 ml of culture medium. Culture of canine bone marrow mesenchymal stem cells: Following MSC isolation, presumptive MSCs were seeded into a 100 mm-Petri dishes (BD-FalconTM, Franklin Lake, NJ, USA), containing low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Invitrogen, Carlsbad, USA), 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. Nonadherent cells were removed by washing the culture dishes with Dulbecco’s phosphate buffered saline (DPBS; Invitrogen) and the culture medium was changed every 2-3 days. Adherent cells were cultured (passage 0) until they reached approximately 70-80% confluence. To sub-passage, the adherent cells were washed twice with DPBS and then digested with 0.125% trypsin-EDTA (GibcoTM, Invitrogen) for 2 min and the enzyme was inactivated with an excessive amount of fetal bovine serum in DMEM. The cell suspension was then centrifuged at 4°C and 1000 rpm for 5 min. If cryopreservation of cells were needed, a freezing medium containing 10% (v/v) dimethylsulphoxide (DMSO) and 90% (v/v) fetal bovine serum was added to the cells. The equilibrated cell suspension was added into a 1-ml cryovial (Corning, USA). The freezing rate was controlled at 1°C/min using a cryobox. Cell morphology and population doubling time: MSCs were daily examined for cell morphology at 100 and 200x magnification using a phase contrast microscope (CKX41, Olympus, Japan). At the 3rd passage, the MSCs were plated into a 12-well plate at 20,000 cells/cm2 (approximately, 40-50% confluence). The MSCs were then trypsinized with trypsin-EDTA, and the total number of cells in each culture well was counted using a hemocytometer at 24 hours interval for 3 consecutive days. The doubling time was calculated using the equation, ln(2)/growth rate, whereas the growth rate referred to the number of doublings that occurred per unit of time. Flow cytometry analysis: Canine MSCs at the 3rd passage were immunologically examined for surface markers of MSCs. Because there is no universal marker that is specific to MSCs, identification of MSCs therefore relied on both positive and negative markers. To perform flow cytometry, the MSCs were first dissociated from the Petri dishes with TrypsinEDTA and then centrifuged. A total of 200,000 to 300,000 cells were stained with each respective antibody. Rat monoclonal anti-canine CD 34 conjugated with fluorescein isothiocyanate (FITC) (a marker for hemopoietic stem cells) was used as the negative MSC marker. Rat monoclonal anti-canine CD 90 (AbD serotec, Kidlington, UK) with rabbit anti-rat FITC secondary antibody and monoclonal anti-canine CD44 conjugated with allophycocyanin (APC) (R&D system, Minneapolis, USA) were used as MSC positive markers. Fluorescently-labeled MSCs were finally washed once, fixed with 1% (w/v) paraformaldehyde in PBS and stored in the dark at 4°C until analysis. Non-staining MSCs and MSCs labeled with only the secondary antibody were used as controls. At least 20,000 MSCs were used to test the
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presence of each cell surface marker, using flow cytometry (BD Biosciences, Franklin Lakes, USA). Differentiation of MSCs: The MSCs derived from the canine bone marrow at the 3rd-5th passages from 2 dogs were used to demonstrate their differentiation potentials. MSCs were induced to differentiate into bone, cartilage and adipose tissues according to the methods as previously described (Bosch et al., 2006), with some modifications. For bone differentiation, MSCs were first sub-cultured to reach approximately 80% confluence, and the bone induction medium consisting of DMEM supplemented with 10% (v/v) FCS, 100 nM dexamethasone, 50 ng/ml ascorbic acid and 10 mM Beta-glycerophosphate was added into the Petri dishes. The bone induction medium was changed every 2-3 days. A three-dimensional culture system was used to induce cartilage differentiation. MSCs were dissociated and then transferred to a 15ml cornical tube (BD-FalconTM, Franklin Lake, NJ, USA). After centrifugation, aggregated cells were resuspended with the cartilage induction medium containing DMEM, 10 ng/ml TGF-β1, 100 nM dexamethasone and 50 ng/ml ascorbic acid 2phosphate. The medium was changed every 2-3 days. The adipose tissue differentiation was performed with monolayer MSCs, as previously described (Zuk et al., 2002). The MSCs were treated with 4 cycles of adipogenic induction and maintenance (each cycle consisted of 3 days of adipogenic induction and 2 days of adipogenic maintenance). The adipogenic induction medium consisted of DMEM 10% (v/v) FCS, 0.1 mg/ml human recombinant insulin, 10 mM sodium pyruvate, 1 mM methyl isobutylxanthine (IBMX), 0.2 mM indomethacin and 1 µM dexamethasone. Adipogenic maintenance was prepared in a similar manner to adipogenic induction, but without IBMX, dexamethasone and indomethacin. Neurogenic induction was performed with DMEM supplemented with 200 µM butylated hydroxyanisole, 25 mM KCl, 2mM valproic acid, 10 µM forskolin, 1 µM hydrocortisone, 5 µg/ml Insulin and 2% (v/v) DMSO. Assessment of MSC differentiation: After the fixation of MSC-derived bone tissues in 4% (w/v) paraformaldehyde, the tissues were embedded in paraffin for histological analysis. The histological sections of the tissues were stained with Von Kossa and alcian blue to detect the deposition of calcium phosphate and glycosaminoglycans enriched-matrix, indicating the successful differentiation of MSCs into bone and cartilage tissues, respectively. The presence of adipogenesis was confirmed by oil red O staining which detects intracellular neutral triglycerides and lipids.
Reverse transcription polymerase chain reaction (RTPCR): Total RNA was extracted from undifferentiated MSCs that were cultured in monolayer and differentiated cell pellets collected on D9 and D21 of differentiation using Absolutely RNA® Nanoprep Kit (Stratagene, Agilent Technologies, USA). The contaminating genomic DNA was removed during the purification steps by DNase I treatment according to the manufacturer’s instructions. Total RNA was eluted from the purification column with sterile distilled water and was quantified using a NanoDrop® 1000 spectophotometer (Thermo Scientific, USA). Gene-specific oligonucleotide primers were synthesized by BioDesign Co., Ltd. (Bangkok, Thailand). Two target genes reported to be involved in osteogenic lineage differentiation, i.e., collagen type I alpha I (COL1A1) and bone γcarboxyglutamate protein (GLA), were selected for this experiment. A housekeeping gene, i.e., glyceraldehype 3-phosphate dehydrogenase (GAPDH), was included as the internal control and reference. Oligonucleotide sequences, PCR product size, Genebank accession number and references of primers are shown in Table 1. cDNA was synthesized from an aliquot of 150 to 200 ng of total RNA using random hexamer primers and Omniscript® Reverse Transcription Kit (Qiagen, Hilden, Germany). One microliter of RT reaction was mixed with 12.5 µl of GoTaq® Green Master Mix (Promega, WI, USA), 6.25 µM each of forward- and reverse primers, and sterile distilled water to reach the final volume of 25 µl. PCR was carried out on an Amplitronyx™ thermocycler machine (Nyxtechnik, CA, USA). The suitable cycle condition was determined and applied for each primer pair as follows: GAPDH, 94oC for 2 min, followed by 30 cycles of 94oC for 30 sec, 55oC for 30 sec, 72oC for 45 sec, plus the final extension at 72oC for 5 min; COL1A1 and GLA, 94oC for 1 min, followed by 30 cycles of 94oC for 30 sec, 58oC for 30 sec, 72oC for 1 min, plus the final extension at 72oC for 10 min. PCR products were analyzed by electrophoresis on 2% ethidium bromide incorporated-agarose gels in TrisBoric-EDTA (TBE) buffer. The images of agarose gels were taken using GeneFlash Gel Documentation (Syngene, Synoptic Ltd., Frederick, USA) and the calculation of relative intensity of target gene mRNA expression signal utilizing the expression intensity of GAPDH as the reference was carried out using the Scion Imaging program (Beta 4.0.3; Scion Corporation, MD).
Table 1 Oligonucleotide primers used in this study Gene COL1A1 GLA GAPDH
Tharasanit T. et al. / Thai J. Vet. Med. 2011. 41(1): 79-86.
Results Fibroblastic-like cells were isolated from canine bone marrow (Fig. 1). These cells adhered to the culture dish by 24 hours of culture. At passage 3, putative MSCs were cultured and analyzed for expression of MSC surface markers and population doubling time. These MSCs were expressed mesenchymal stem cells markers (CD 44 and CD 90) and also negative to hemopoietic stem cells marker (CD 34). Two canine MSCs (dog 3 and dog 4) strongly expressed CD 44 (99.9% and 86.7%) and CD 90 (92.5%, and 95.3%), respectively (Fig. 2). However, the growth rates of MSCs (population doubling time) were found to be different among putative MSCs from different dogs (46.1, 24.2, 38.8 and 32.4 hours). We further demonstrated that these cells were also capable of differentiation into mesodermal lineages including bone, cartilage and adipose tissues. After 21 days of differentiation, differentiated MSCs were essentially positive to oil red O, Von kossa and alcian blue as indicators of fat, bone and cartilage formation respectively (Fig. 3). In addition, MSCs were also transdifferentiated into multiple-process neuron-like cells soon after treating MSCs with neurogenic medium (Fig. 3).
Figure 1 Fibroblastic-like mesenchymal stem cells derived from bone marrow aspirate
COL1A1 and GLA expression was detected in undifferentiated MSCs. The early stage of differentiation up-regulated the genes as seen in the increment of relative signal intensity on D9 post culture. COL1A1 mRNA increased continuously on D21 post differentiation, while the expression of GLA gene dropped below the starting point (Fig. 4).
Figure 2 Expressions of CD 44 and CD 90 in canine MSCs. Samples from dog 3 (upper panel) and dog 4 (lower panel) were analyzed by flow cytometry.
Tharasanit T. et al. / Thai J Vet Med. 2011. 41(1): 79-86.
Figure 4 Relative signal intensity of COL1A1 and GLA expression compared with GAPDH expression on D0, D9 and D21 of dog MSCs bone differentiation in vitro.
Figure 3 Differentiation potential of canine mesenchymal stem cell (A) adipose tissue; cartilage (B) bone (C and E) and neuron-like cells (F). Adipocytic cells accumulated with neutral lipids as shown by oil red O staining. Cartilagenous and bone tissues were confirmed by the presence of glucosaminoglycan enriched matrix, calcium phosphate and alkaline phosphatase activity (D) using alcian blue, Von kossa and leukocyte alkaline phosphatase kit, respectively.
Discussion In the current study, we successfully isolated well-defined mesenchymal stem cells derived from canine bone marrow. These specialized cells are classified as multipotent stem cells because they have the capability of differentiation into mesodermal lineage. MSCs have been isolated from many tissues of the body such as bone marrow (Wagner et al., 2005; Kern et al., 2006), adipose tissue (Zuk et al., 2002), umbilical cord (Lee et al., 2004; Koch et al., 2007 ) and dental pulp (Jo et al., 2007; Waddington et al., 2009). In dog, the data in regard to the isolation, characterization and clinical use of canine MSCs have been limited. Until recently, canine MSCs have been isolated from bone marrow (Csaki et al., 2007; Jafarian et al., 2008), adipose tissue (Neupane et al., 2008; Vieira et al., 2010) and umbilical cord (Seo et al., 2009). Because there is no specific marker for the identification of canine MSCs, the isolation and identification technique has therefore been different among laboratories. MSCs from many species demonstrate antigen specific on cell membrane such as CD 29, CD 44, CD 90, CD 105 and Stro-1 (Martin et al., 2002; Bosnakovski et al., 2005; Csaki et al., 2007; Meirelles and Nardi, 2009; Rho et al., 2009). In the current study, only canine antibodies were proven a good candidate for MSC isolation when compared with antibodies from other animals such as anti-mouse Stro-1 and anti-human CD 105 (unpublished data), suggesting
the specificity of canine surface antigen. This phenomenon is one of the hallmarks affecting the exploitation of MSCs for clinical use since MSCs are located in the bone marrow with a mixed population of cells and it has been estimated that there are only 0.0001-0.01% MSCs in the nucleated cells of bone marrow aspirate (Pittenger et al., 1999). Most investigators have isolated MSC using their capacity to adhere to a plastic culture dish. However, macrophages, endothelial cells, lymphocytes, and smooth muscle cells can also adhere to culture plate and therefore contaminate the MSC preparations. The isolation, identification and purification of canine MSCs recently become an important issue for the clinical use of MSCs. Although legal regulation use of canine MSCs has yet to be discussed, the minimum requirements for clinical use of MSCs have been announced for human (Dominici et al., 2006). Human MSCs must demonstrate the fibroblast-like morphology and have the ability to adhere to a plastic culture dish, positively express (> 95%) cell surface receptors (e.g. CD 29, CD 44, CD 73, CD 105, CD 106, Stro-1, etc.) and negatively express (< 2%) the hematopoietic lineage markers (e.g. c-Kit, CD 14, CD 34, CD 45). More importantly, these cells must show a capability of differentiation into mesodermal lineage (bone, cartilage and adipose tissues). In the current study, we found that MSCs, at least under our conditions, were already committed to differentiate into bone and cartilage as they prematurely expressed GLA and COL1A1 (early markers for bone and cartilage differentiation) prior to differentiation induction. It is also possible that the MSCs used in this study had spontaneous differentiation during culture. Until recently, study of pathways regulating the bone and cartilage differentiation of canine bone marrow MSCs is still required in order to improve the efficiency of in vitro bone and cartilage differentiation for cell- or tissue-based engineering. Volk et al. (2005) reported that canine MSCs could be efficiently differentiated into bone using bone morphologic protein 2 (BMP-2) which was similar to other species (Li et al., 2007). In addition to the differentiation capability into mesodermal lineage, transdifferentiation of MSCs to neuron-like cells has been believed to hold a great promise in cell treatment therapy (Zipori, 2004; Krabbe et al., 2005; Bongso et
Tharasanit T. et al. / Thai J Vet Med. 2011. 41(1): 79-86. al., 2008). However, this transdifferentiation of MSCs into neuron-like cells has been contradictory especially the toxic effect of DMSO on actin microfilament during differentiation (Lu et al., 2004; Neuhuber et al., 2004). Interestingly, it has been shown that MSCs expressed neuronal specific genes even though they were not treated with neuronal differentiation medium (Yamaguchi et al., 2006; Kamishina et al., 2006). In this study, we only recorded morphological changes of canine MSCs after neuronal differentiation. The ‘true’ capability of canine MSCs in neuron differentiation has yet to be examined in the prospective study. In conclusion, our study demonstrates that canine mesenchymal stem cells can be isolated from bone marrow, and these MSCs are capable of differentiation into specific mesodermal linage (bone, cartilage and adipose tissue) following passages and in vitro differentiation.
Acknowledgement This work was financially supported by the Innovation Center for Veterinary Science, Faculty of Veterinary Science, Chulalongkorn University, Chulalongkorn University Centenary Academic Development Project, the Thailand Research Fund MRG5380153, TRF-DBG5280015 and TRF Senior Research Scholars RTA-5080010.
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Anticlastogenic Effect of Asiatic Pennywort and Indian Mulberry using Rodent Erythrocyte Micronucleus Assay Piengchai Kupradinun1* Anong Tepsuwan2 Wannee R. Kusamran2†
Abstract Asiatic pennywort [Centella asiatica (L.), Urban, AP] and Indian mulberry (Morinda citrifolia Linn., IM), the Asian plants, have been previously demonstrated in our laboratory that they could markedly enhance the activities of some phase II enzymes, but significantly decreased the activities of phase I enzymes, indicating that they may possess cancer chemopreventive potentials. This study was to investigate the anticlastogenic activity of AP and IM against a direct-acting clastogen, mitomycin C (MMC), and indirect-acting clastogens, cyclophosphamide (CYP) and 7, 12dimethylbenz(a)anthracene (DMBA). Male mice were fed with AP or IM leaves mixing in modified AIN -76 semipurified diet, or IM fruit juice or fruit powder solution by gavage, for 2 weeks prior to administration of clastogens. Anticlastogenic effect was determined by using in vivo erythrocytes micronucleus assay. Blood samples were collected and counted for reticulocytes with and without a micronucleus using the fluorescent microscope. After comparing with the controls, we found that 25% of AP and IM leaves in diets significant decrease micronucleated peripheral reticulocytes (MNRETs) induced by MMC, CYP and MMC, DMBA, respectively (p<0.05). While IM fruit juice at 10 and 20 ml/kg BW caused a significant decrease in MNRETs induced only by MMC. IM fruit powder at 100 and 500 mg/kg BW decreased MNRETs induced only by MMC in a dose dependent manner, however the significant decrease was found only in the high dose (p<0.05). These results demonstrated that AP leaves and IM (leaves and fruits) were able to inhibit the clastogenic activity of both direct and indirect-acting clastogens in the mouse, particularly IM leaves which showed the highest inhibitory effect. Keywords: anticlastogenic, Asiatic pennywort, Indian mulberry, micronucleus, MNRETs, mouse Laboratory Animal Section, 2Biochemistry and Chemical Carcinogenesis Section, Research Division, National Cancer Institute, Ministry of Public Health, Bangkok 10400, Thailand †Dedicated to the memory of Dr. Wannee R. Kusamran deceased on Jan 1, 2011. Corresponding author E-mail: [email protected]ahoo.com 1
Thai J Vet Med. 2011. 41(1): 87-94.
Kupradinun, P. et al. Thai J Vet Med. 2011. 41(1): 87-94.
บทคัดย่อ ผลการยับยั้งการเกิดไมโครนิวเคลียสของบัวบก และยอในเม็ดเลือดแดงของหนูทดลอง เพียงใจ คูประดินันท์ 1* อนงค์ เทพสุวรรณ 2 วรรณี คูสําราญ 2† บัวบก [Centella asiatica (L.), Urban] และยอ (Morinda citrifolia Linn.) เป็นพืชประจําท้องถิ่นในเอเชีย งานวิจัยของ สถาบันมะเร็งแห่งชาติที่ผ่านมาพบว่า พืชเหล่านี้สามารถกระตุ้นเอนไซม์ในกระบวนการ biotransformation ใน Phase II แต่ยับยั้งเอนไซม์ ใน phase I ซึ่งแสดงว่าพืชเหล่านี้มีสารเคมีที่มีศักยภาพในการป้องกันมะเร็ง การศึกษานี้มีจุดประสงค์เพื่อ ตรวจสอบการยับยั้งการเกิดไมโคร นิวเคลียสของเม็ดเลือดแดงที่ชักนําด้วยสารก่อมะเร็งที่ออกฤทธิ์โดยตรง (Mitomycin C, MMC) และชนิดที่ออกฤทธิ์ทางอ้อม (Cyclophosphamide, CYP และ 7,12-dimethylbenz(a)anthracene, DMBA) โดยการให้หนูเม้าส์เพศผู้ กินใบบัวบก และยอ (ใบและ ผล) ที่ผสมในอาหารผงหรือโดยการป้อนทางปาก เป็นเวลา 2 สัปดาห์ก่อนให้สารก่อมะเร็ง ตรวจสอบการยับยั้งการเกิดไมโครนิวเคลียสด้วย วิธี in vivo erythrocyte micronucleus assay ผลการศึกษาพบว่าใบบัวบกที่ผสมในอาหารร้อยละ 25 สามารถลดจํานวนเม็ดเลือดแดงที่มี ไมโครนิวเคลียส (MNRETs) ที่ชักนําด้วย MMC และ CYP อย่างมีนัยสําคัญทางสถิติเมื่อเทียบกับกลุ่มควบคุม (p<0.05) ใบยอในขนาดร้อยละ 25 ในอาหารสามารถลดจํานวน MNRETs ที่ชักนําด้วย MMC และ DMBA ได้อย่างมีนัยสําคัญทางสถิติ (p<0.05) น้ําลูกยอในขนาด 10 และ 20 มล./นน.ตัว 1 กก. สามารถลดจํานวน MNRETs ที่ชักนําด้วย MMC เท่านั้นอย่างมีนัยสําคัญทางสถิติ (p<0.05) ส่วนผงลูกยอในขนาด 100 และ 500 มก./นน.ตัว 1 กก. สามารถลดจํานวน MNRETs ที่ชักนําด้วย MMC ได้ตามขนาดที่เพิ่มขึ้น อย่างไรก็ตามพบความแตกต่าง อย่างมีนัยสําคัญทางสถิติเฉพาะในขนาดสูงเท่านั้น (p<0.05) ผลการศึกษานี้แสดงว่าใบบัวบกและยอ (ใบ และผล) มีฤทธิ์ยับยั้งการเกิดไมโคร นิวเคลียสที่ชักนําด้วยสารก่อมะเร็งทั้งชนิดที่ออกฤทธิ์โดยตรงและ ชนิดที่ออกฤทธิ์ทางอ้อมในหนูเม้าส์ โดยเฉพาะใบยอมีฤทธิ์ในการยับยั้งสูง ที่สุด คําสําคัญ: ใบบัวบก ใบยอ ลูกยอ ไมโครนิวเคลียส หนูเม้าส์ 1 งานสัตว์ทดลอง 2 งานชีวเคมีและวิจัยสารก่อมะเร็ง กลุ่มงานวิจัย สถาบันมะเร็งแห่งชาติ กระทรวงสาธารณสุข กรุงเทพฯ 10400 † เป็นเกียรติแด่ ดร. วรรณี คูสาํ ราญ เสียชีวิตเมื่อ 1 มกราคม 2554 *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction It has been estimated that more than twothirds of human cancers can be prevented through appropriate lifestyle modification (Doll et al., 1981). The risk of some cancers may be reduced by consumption of various kinds of vegetables and fruits (Block et al., 1992). A number of vegetables and fruits contain various kinds of chemicals possessing chemopreventive potentials (Wattenberg, 1985). Chemopreventive agents may function by a variety of mechanisms such as directed at all major stages of carcinogenesis (Wattenberg, 1997); induction of phase II detoxification enzymes; the inhibition of phase I activating enzymes, as well as the inhibition of the mutagenicity/clastogenicity of chemical carcinogens (Talalay, 1992). We have previously reported that some vegetables, for examples, neem flowers, sesbania flowers, Thai and Chinese bitter gourd fruits, leaves
of sweet basil, Siamese cassia, ivy gourd, lemon grass and Indian mulberry, possess antimutagenic activity towards Salmonella typhimurium (Rojanapo et al., 1993; Kusamran et al., 1998a). Some of these vegetables including neem flowers, Asiatic pennywort leaves, Cassia leaves, Thai bitter gourd fruits, kale and Indian mulberry leaves can also increase the activity of some phase II detoxification enzymes while decreasing the activity of some phase I enzymes (Tepsuwan et al., 1997; Kusamran et al., 1998b). In addition, some of them can inhibit the clastogenicity of the clastogens in mice (Kupradinun et al., 1997; Kupradinun 2008) and inhibit some chemically induced carcinogenesis in rats (Kusamran et al., 1998a; Tepsuwan et al., 1999; 2002). Asiatic pennywort (AP) is a tropical plant which is used for health supplement. This plant possesses biological activities such as antioxidation (Jayashree et al., 2003) and chemopreventive property against azoxymethane-induced aberrant crypt foci (ACF) formation in rat colon (Bunpo et al., 2004) and
Kupradinun, P. et al. Thai J Vet Med. 2011. 41(1): 87-94. induction of cell cycle arrest in colon cancer cell line (Caco-2 cells) (Bunpo et al., 2005). Indian mulberry (IM) leaves are commonly used in Thai cuisine while fruit juice is widely consumed as it has been claimed to possess anti-inflammatory and antitumor properties (Hirazumi et al., 1994; Hirazumi and Furusawa, 1999; Wang and Su, 2001; Furusawa et al., 2003). The in vivo rodent micronucleus assay in bone marrow cells has been accepted for evaluation of the clastogenicity of chemical compounds (Matter and Schmid, 1973; Schmid, 1975). In 1980, using mouse peripheral blood instead of bone marrow cells was introduced for use in the micronucleus assay (MacGregor et al., 1980) and acridine orange has been used as supravital staining of blood cells (Hayashi et al., 1983). These modifications offer many advantages to conventional bone marrow assay and widely used to evaluate chemical clastogenicity. This technique has also been shown to be very useful as a short-term assay for evaluating the anticlastogenicity as well as chemopreventive potential of compounds (Heo et al., 1996; Kupradinun et al., 1997; Kupradinun, 2008; Hwan et al., 2008; Promkum et al., 2010). In this study, we applied this technique to determine the anticlastogenicity of AP and IM which have been demonstrated to possess both antimutagenicity (Kusamran et al., 1998a) as well as chemopreventive potentials (Bunpo et al., 2004) against both direct-acting (mitomycin C, MMC) and indirect-acting clastogens (cyclophosphamide, CYP and/or DMBA). Anticlastogenic effect of AP and IM demonstrated in this study would lead to further research on its mechanism or modulating factors related to these effects. Moreover, it will give information whether this technique can be used to predict the chemopreventive potential of chemical compound in vegetables and medicinal plants.
Materials and Methods Chemicals: MMC, a direct-acting clastogen was purchased from Kyowa Hakko Kogyo Co. (Tokyo, Japan) while CYP and DMBA (indirect-acting clastogens) were obtained from ASTA Medica AG (Frankfurt am Main, Germany) and Sigma Chemicals (St. Louis, USA), respectively. All vitamins used for the preparation of vitamin mixture were obtained from Sigma Chemicals Co. (St. Louis, USA). Acridine orange (AO) was obtained from E. Merk (Germany). Chemicals used for the preparation of salt mixture were obtained from Fluka Chemicals Co. (Switzerland) and casein (EM HV milk protein) was the product of D.M.V. Co. (The Netherland). Plant preparations: AP and IM leaves were purchased from local markets in Bangkok. They were washed with tap and distilled water, chopped into small pieces and then lyophilized. Freeze-dried vegetables were blended to powder and kept at -20oC until use. IM fruit juice was purchased from a supermarket in Bangkok while IM fruit powder was obtained from Abhaibhubejhr Hospital, Thailand. IM
89 fruit powder was dissolved in distilled water under 40o-50oC before giving to the animals. Animals and diets preparation: A total of 270 male ICR mice, 5 weeks old, aged 27±3 g, were obtained from the National Laboratory Animal Center, Mahidol University, Thailand. Animals were maintained at the Laboratory Animal Facility of the National Cancer Institute according to the Institutional Care Guidelines, which were approved by the Animal Ethic Committee. All animals were housed in shoes box stainless steel cages in an airconditioned room at 23o±2oC and relative humidity 50±20% with 12 hours light/dark cycle. For each experiment, animals were acclimatized for 5-7 days by giving a modified AIN-76 semi-purified diet (basal diet) according to Bieri et al., 1997 and Reeves et al., 1993 or pellet diet (Perfect Companion Co. Ltd., Thailand) before starting the experiment. The vegetable diets were prepared by substituting the ground freeze-dried AP or IM leaves at 12.5% and 25% in a modified AIN-76 diets as previously described (Kusamran et al., 1998a). Clastogenicity testings After acclimation, the animals were randomly divided by weight into 3 groups of 8-10 mice each. One group was assigned as control group that continued to receive the basal or pellet diets, while the other two groups were assigned as experimental groups receiving basal diet containing low (12.5%) and high (25%) doses of ground freezedried AP/IM or 2 doses (10 and 20 ml/kg BW, approximately equivalent to 5 and 10 times of human consumption) of IM fruit juice and 100 and 500 mg/kg BW of IM fruit powder solutions (approximately equivalent to and 5 times of human consumption) daily by gavage for 2 weeks and continued till the end of the experiment. In groups receiving vegetable diets, both control and experimental groups were pair-fed and water ad libitum. For IM fruit juice and powder groups, the mice were given a pellet diet and the control group were given vehicle and distilled water, respectively by gavage. At 2 weeks after feeding the experimental diets or samples, blood samples were collected and subjected to micronucleus assay (Fig. 1) as previously described (Kupradinun, 2008). Briefly, 5-7 µl of mouse peripheral blood were placed on AO-coated glass slides and covered with 22x40 mm cover slip. After few hours, micronucleated peripheral reticulocytes (MNRETs) types I, II, and III as classified by Vander et al. (Vander et al., 1963) were counted under the fluorescent microscope (Olympus Model BH-2, Japan). The frequencies of MNRETs were recorded based on the observation of all 1000 reticulocytes per mouse. Anticlastogenicity testings A. Anticlastogenicity testing against MMC MMC was intraperitoneally injected into mice that have been used previously for clastogenicity test at 1 mg/kg BW just after blood samples were collected. Then blood samples were collected at 24
Kupradinun, P. et al. Thai J Vet Med. 2011. 41(1): 87-94.
and 48 hours after MMC injection and micronucleus formation were analyzed (Fig. 1). B.
Anticlastogenicity testing against CYP and/or DMBA
Two out of the 3 experimental groups for clastogenicity study were administered with CYP at 50 mg/kg BW (i.p) or DMBA at 40 mg/kg BW (in corn oil, p.o). Blood samples were collected at 24 and 48 hours after clastogens administration and analyzed for reticulocytes as in an experiment A (Fig. 1).
Figure 1 Schematic diagram of experimental design. Open bar was control group (Gr. 1). Dot bar and hatched bar were experimental groups (Grs. 2,3). Arrows: blood collection. Black arrows: clastogens: mitomycin C (MMC); cyclophosphamide (CYP); 7,12-dimethylbenz(a)anthracene (DMBA).
Statistical analysis: Significant difference in the frequencies of MNRETs between the experimental and control groups was analyzed using KruskalWallis H and nonparametric Mann-Whitney U tests at p<0.05.
Results Effect of AP and IM on the body weight and food consumption: Body weight and food consumption were daily recorded for the entire experiment. There were no significant differences in body weight and food consumption between the control and experimental groups (data not shown). Effect of AP leaves on the micronucleus formation in mice peripheral blood: The effects of AP leaves on the micronucleus formation in male mice treated with a direct-acting clastogen, MMC and indirect-acting clastogens, CYP, and DMBA are shown in Fig. 2. The number of MNRETs in the control and experimental groups were not significantly different. The results showed that the frequency of MNRETs of mice in all groups increased with time-course and reached the maximum number at 48 hours after MMC treatment (Fig. 2A). Therefore, the percent inhibition was calculated to compare the inhibitory effect of these plants against clastogens induced MNRETs at 48 hours after treatment. MNRETs in mice fed with AP leaves decreased at 24 hours and 48 hours in both low and high dose groups, but were significantly different (p=0.049) from those of the control group only a high dose at 48 hours (Fig. 2A). Figure 2B shows MNRETs in mice induced by CYP. MNRETs decreased at 24 and 48 hours in mice fed with AP leaves at both low and high doses. The significant differences (p=0.002) from those of the control were recorded only in high dose group at both time points. While in low dose group, MNRETs significantly decreased only at 24 hours. The inhibitory effects at high and low doses of AP diets were 44% and 10%, respectively. Figure 2C shows MNRETs in mice induced by DMBA. It was found that the feeding of AP leaves resulted in the increase of MNRETs in the low dose group at both time points, which was significantly different (p= 0.045) from those of the control group at 24 hours. However, MNRETs of the high dose group decreased at both time points, but were not significantly different from those of the control group.
Figure 2 Mean frequencies of MNRETs in mice given AP leaves after clastogens administration. The mean frequencies of MNRETs in the control group received basal diet (open bar) and the experimental groups (dot bar and hatched bar) received ground freeze-dried AP leaves at 12.5% and 25% in the diets, respectively. * Significant differences at p<0.05.
Kupradinun, P. et al. Thai J Vet Med. 2011. 41(1): 87-94.
Figure 3 Mean frequencies of MNRETs in mice given IM leaves after clastogens administration. The mean frequencies of MNRETs in the control group received basal diet (open bar) and the experimental groups received ground freeze-dried IM leaves at 12.5% (dot bar) and 25% (hatched bar) in the diets. * Significant differences at p<0.05.
Figure 4 Mean frequencies of MNRETs in mice given with IM fruit juice after clastogens administration. The mean frequencies of MNRETs in the control group which received vehicle (open bar) and the experimental groups received IM fruit juice at 10 (dot bar) and 20 ml/kg BW (hatched bar) by gavage. * Significant differences at p<0.05.
Figure 5 Mean frequencies of MNRETs in mice given with IM fruit powder solutions after clastogens administration. The mean frequencies of MNRETs in the control group which received distilled water (open bar) and the experimental groups received IM fruit powder solutions at 100 (dot bar) and 500 mg/kg BW (hatched bar) by gavage. * Significant differences at p<0.05.
Effect of IM leaves and fruits on the micronucleus formation in mice peripheral blood: The number of MNRETs in the control and experimental groups were not significantly different. IM leaves at 12.5% in the diet did not affect MNRETs induced by MMC (Fig 3A). However, IM leaves at 25% in the diet significantly decreased MNRETs (p=0.05) at 48 hours with 53% inhibition. IM leaves slightly increased MNRETs induced by DMBA at 24 hours in both low or high dose groups (Fig. 3B). On the contrary, at 48 hours MNRETs decreased in both low and high dose groups and were statistically significant (p=0.047)
only at the high dose with 53% inhibition. In mice fed with IM fruit juice, we found that of the dose at 10 and 20 ml/kg BW decreased MNRETs induced by MMC at 24 and 48 hours (Fig. 4A), particularly MNRETs was significantly different from those of the control group at the low dose (p<0.05), However, in the high dose group, MNRETs was significantly different (p=0.018) only at 48 hours. The inhibitory effects at both low and high doses were quite similar about 40-46%. While in DMBA induced mice in Fig. 4B, MNRETs slightly increased at 48 hours in the low dose group, but slightly decreased in
Kupradinun, P. et al. Thai J Vet Med. 2011. 41(1): 87-94.
the high dose group. Figure 5A illustrates the mean frequency of MNRETs in IM fruit powder treated groups induced by MMC. IM fruit powder shows spontaneous micronucleus formation at low dose (100 mg/kg BW). MNRETs decreased in both low and high dose groups (100 and 500 mg/kg BW) in a dose dependent manner. However, the reduction was statistically significant (p=0.028 and p=0.016), respectively, only in the high dose group at both 24 and 48 hours. The inhibitory effect at high dose was nearly twice higher than at low dose (60% VS 39%). In IM fruit powder treated group induced by DMBA (Fig. 5B), MNRETs either in the low or high dose group slightly decreased at both time points.
Discussion According to pair feeding of mice in this study, e.g. leaves of Asiatic pennywort and Indian mulberry, including oral administration of Indian mulberry fruits had no effect on the body weight of the mice. AP leaves, IM leaves and fruit juice except IM fruit powder had no effect on the spontaneous formation of MNRETs in the mouse. It means that Indian mulberry fruit powder had both clastogenic and anticlastogenic activities. The criterion for evaluation of a positive result is a statistically significance with dose-related increase in frequency of micronucleated erythrocyte at any time point with at least 1 value significantly exceeding the vehicle control (Hayashi et al., 1994). In this study all plants significantly reduced MNRETs induced by MMC and CYP but they did not significantly reduce MNRETs induced by DMBA, except IM leaves. It was possible that the different mechanism of action might be used to suppress the micronucleus formation induced by these clastogens (Iyer and Szybaloki, 1964; Erlichman, 1992). The results of the present study demonstrated that AP leaves and IM (leaves and fruits) had significant effect on the micronucleus formation especially in the high dose. AP leaves at low concentration in the diet increased MNRETs induced by DMBA at both time points. On the contrary, AP leaves at high concentration could reduce MNRETs. It was opposite to the results of Bunpo et al. (2004) which used water extract of AP on AOM-induced ACF formation in rats and found less effective at a high dose. It might be the differences in pharmacokinetic of chemical constituents in this plant such as glycoside, asiatic acid, asiaticoside, medecassic acid and meecassoside (Grimaldi et al., 1990). In addition, our previous study showed that anticarcinogenic potential of AP leaves was uncertain because they caused both induction and reduction of phase I enzymes and also induction of phase II enzymes in rats (Tepsuwan and Kusamran, 1997). IM leaves had the most potent chemopreventive properties especially at high dose in the diets possess anticlastogenic activity against both
direct and indirect-acting clastogens. Its anticlastogenic potential might be modulated via direct acting carcinogenesis related with DNA-crosslinking agents (Iyer and Szybaloki, 1964) and via indirect carcinogenesis, which require metabolic activation to become a highly reactive metabolite and bind to DNA forming labile covalent DNA adducts in order to become mutagenic and/or carcinogenic (Erlichman, 1992). It is correlated with our recent research that IM leaves contain compounds acting as a monofunctional inducer and phase I inhibitor in the rats (Tepsuwan and Kusamran, 1997). In addition, IM leaf extracts have shown antimutagenic activities against an indirect-acting mutagen (Kusamran et al., 1998a). IM fruits, both juice and powder at the concentration tested can inhibit the clastogenicity of only a direct-acting clastogen. However, IM fruit powder had both clastogenic activity and more pronounced chemopreventive effect which we should further study. Recent research of IM fruit juice have shown antitumor activity against lung cancer in C57BL/6 mice (Hirazumi et al., 1994). In addition, IM fruit juice has antioxidant activity (Wang and Su, 2001) and cancer preventive effect at the initiation stage of carcinogenesis (Wang et al., 2002). There are possibilities that many phytochemicals found in IM fruit juice as well as vitamins and minerals can suppress tumor growth by stimulating the immune system. However, its chemopreventive properties are not clear at the moment (Potterat et al., 2007).
Conclusion We concluded that Asiatic pennywort and Indian mulberry contained some anticlastogens, indicating that they might have chemopreventive potential against genotoxicants. Among the 2 vegetables, IM leaves showed the highest chemopreventive potential. The inhibitory effect of these vegetables seemed to correlate quite well with the capacity to enhance phase II enzyme activity and to decrease phase I enzyme activity as well as acting as the blocking agents. Micronucleus assay could be used as screening method for detecting chemopreventive agents.
Acknowledgement This study was financially supported by a grant from the National Cancer Institute, Ministry of Public Health, Thailand. We thank Ms. Sarochin Kunrach and Mr. Chainarong Thongoen for their excellent technical assistance.
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94 leaves of Siamese cassia, Indian mulberry and Asiatic pennywort on the metabolizing enzymes of chemical carcinogens in rat liver. Bull Dept Med Serv. 22: 425-437. Tepsuwan, A., Kupradinun, P. and Kusamran, W.R. 1999. Effect of Siamese cassia leaves on the activities of chemical carcinogen metabolizing enzymes and on mammary gland carcinogenesis in the rat. Mutat Res. 428: 368-373. Tepsuwan, A., Kupradinun, P. and Kusamran, W.R. 2002. Chemopreventive potential of neem flowers on carcinogen-induced rat mammary and liver carcinogenesis. Asian Pac J Cancer Prev. 3(3): 231-238. Vander, B.J., Harris, A.C. and Ellis, R.S. 1963. Reticulocyte counts by means of fluorescence microscopy. J Lab Clin Med. 62: 132-140. Wang, M.Y. and Su, C. 2001. Cancer preventive effect of Morinda citrifolia (Noni). Ann N Y Acad Sci. 952: 161-168. Wang, M.Y., Anderson, G. and Nowicki, D. 2002. Preventive effect of Morinda citrifolia (noni) at the initiation stage of mammary breast cancer induced by 7, 12-dimethylbenz(a)anthracene (DMBA) in female SpragueDawley rats. The Proceedings of the Frontiers in cancer prevention research, AACR, Boston, Oct 17. Wattenberg, L.W. 1985. Chemoprevention of cancer. Cancer Res. 45: 1-8. Wattenberg, L.W. 1997. Inhibition of carcinogenic effects of polycyclic hydrocarbons by benzyl isothiocyanate and related compounds. J Natl Cancer Inst. 58(2): 395-398.
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Seroprevalence of Antibodies against Bartonella hensalae Infection in Cats and Dogs along the Northern Borders of Thailand Decha Pangjai 1* Soichi Maruyama2 Sumalee Boonma3 Wimol Petkanchanapong1 Wattanapong Wootta1 Pathom Sawanpanyalert1
Abstract Bartonella hensalae is a causative agent of cat scratch disease that has serious zoonotic potential for people. The aim of the present study is to examine seroprevalence of antibodies against B. hensalae in cats and dogs along the northern borders of Thailand in Chiangrai, Maehongson and Nan provinces using the indirect fluorescent antibody test (IFA). A total of 169 serum samples derived from 56 cats and 113 dogs were collected from 3 provinces along the northern borders of Thailand. It was found that the average percentage of positive rate to B. henselae in Chiangrai, Maehongson and Nan provinces was 73.0%, 52.0% and 0.0%, respectively with the cut-off value of 1:32. The seroprevalence of cats from the Thailand-Myanmar border (Chiangrai and Maehongson provinces) was significantly higher than that of cats from the Thailand-Laos PDR border (Nan province) (61.8% vs 0.0%, p<0.001). There was no prevalence of antibodies against B. henselae from dogs in the 3 provinces. Keywords: Bartonella hensalae, cats, dogs, the northern borders of Thailand 1 Department 2 Laboratory
of Medical Sciences, National Institute of Health, Ministry of Public Health, Nonthaburi, Thailand 11000 of Veterinary Public Health, College of Bioresource Sciences, Nihon University Fujisawa, Kanagawa 252-8510,
Emerging Infections Program (IEIP) Thailand MOPH-U.S. CDC Collaboration (TUC) Dept. of Medical Sciences, Bld 2, 1st Floor Ministry of Public Health, Muang, Nonthaburi 11000, Thailand Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 95-98.
Pangjai D. et al. / Thai J Vet Med. 2011. 41(1): 95-98.
บทคัดย่อ ความชุกของแอนติบอดีต่อการติดเชือ้ Bartonella hensalae ในแมวและสุนัขในพืน้ ที่ชายแดน ไทยภาคเหนือ เดชา แปงใจ 1* โซอิชิ มารุยามา 2 สุมาลี บุญมา 3 วิมล เพชรกาญจนพงศ์ 1 วัฒนพงศ์ วุทธา 1 ปฐม สวรรค์ปัญญาเลิศ 1 Bartonella hensalae เป็นสาเหตุของโรค Bartonellosis ซึ่งเป็นโรคติดต่อจากสัตว์สู่คน คณะผู้วิจัยจึงได้ศึกษาหาความชุกของ แอนติบอดีต่อการติดเชื้อนี้ในสัตว์ที่ใกล้ชิดคน ได้แก่ สุนัขและแมว ในพื้นที่ชายแดนภาคเหนือ 3 จังหวัด ได้แก่ จังหวัดเชียงราย แม่ฮ่องสอน และน่าน โดยวิธีทดสอบแบบ indirect fluorescent antibody test (IFA) ในตัวอย่างทั้งสิ้น 169 ตัวอย่าง ซึ่งเป็นตัวอย่างซีรัมแมว 56 ตัวอย่างและซีรัมสุนัขจํานวน 113 ตัวอย่าง ผลของการศึกษาพบว่า ที่เกณฑ์ตัดสินโรค 1:32 ตัวอย่างซีรัมแมวของจังหวัดเชียงรายพบผลบวก ร้อยละ 73 (11/15) จังหวัดแม่ฮ่องสอน พบผลบวกร้อยละ 52 (10/19 ) ส่วนของจังหวัดน่านไม่พบผลบวก ความชุกของแอนติบอดีตอ่ การติด เชื้อในแมวในพื้นที่ชายแดนไทย-พม่า มีอัตราการสัมผัสเชื้อสูงกว่าในแมวในเขตพื้นที่ชายแดนไทย-ลาวอย่างมีนัยสําคัญทางสถิติ (61.8% ต่อ 0.0%, p<0.001) แต่ไม่พบอัตราการติดเชื้อ B. hensalae ในสุนัข คําสําคัญ: Bartonella hensalae แมว สุนัข พื้นที่ชายแดนไทยภาคเหนือ 1 สถาบันวิจัยวิทยาศาสตร์สาธารณสุข กรมวิทยาศาสตร์การแพทย์ กระทรวงสาธารณสุข จ. นนทบุรี 11000 2 Laboratory of Veterinary Public Health, College of Bioresource Sciences, Nihon University Fujisawa, Kanagawa 2528510, Japan 3 International Emerging Infections Program (IEIP) Thailand MOPH-U.S. CDC Collaboration (TUC) กรมวิทยาศาสตร์การแพทย์ กระทรวงสาธารณสุข จ. นนทบุรี 11000 *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction Genus Bartonella is small, gram-negative aerobic bacilli that is difficult to grow in culture. They are found in many different animals. Bartonella species are the cause of several different diseases associated with human disease including B. bacilliformis, B. quintana, B. vinsonii berkhoffii, B. henselae, B. elizabethae, B. grahamii, B. washoensis, B. koehlerae, B. rocha-limaea and B. tamiae (Lamas et al., 2008). Bartonella henselae is a proteobacterium that can cause bacteremia, endocarditis, bacillary angiomatosis and peliosis hepatitis in human (Anderson and Neuman, 1997). It is also the causative agent of cat scratch disease, occuring after a cat bite or scratch. The disease is characterized by lymphadenopathy and fever (Chomel et al., 2004; Inoue et al., 2009). The study was conducted to understand the serological level of the infection of Bartonella hensalae in cats and dogs in the areas along the ThailandMyanmar-Laos PDR borders. The relationship of dogs, cats and prevalence of Bartonella spp. was also
examined in the present study. The data will benefit public health workers and patients who have a history of contact with commensal vectors such as flea and tick. It will be useful for the practice of public health in controlling vector-borne disease. It can also benefit for the management of the regional offices of Disease Prevention and Control in the province along Thailand-Myanmar-Laos PDR borders.
Materials and Methods Sera: A total of 56 pet cats and 113 pet dogs sera were collected from 3 provinces along the ThailandMyanmar border (Maesai district: Chiangrai province, Muang district: Maehongson province) and ThailandLaos PDR border (Chalermprakiat district: Nan province), from February to March in 2008. Before the collection of blood samples, gender, type and age of each cat and dog were recorded. The dogs and cats were restrained and 3 ml of fresh blood from the cephalic, saphenous or femoral veins was aseptically collected and placed in vials. Sera were separated and stored at -20oC until tested.
Pangjai D. et al. / Thai J Vet Med. 2011. 41(1): 95-98.
Table 1 The gender and age of cats and dogs samples along the northern borders of Thailand Thailand-Myanmar border Species
Chiangrai province Male/Female
Thailand-Lao PDR border
Maehongson province Male/Female
*Young: Cat/Dog age < 1 year **Adult: Cat/Dog age ≥ 1 year
Serological test: Anti-B. henselae detection was performed in the Laboratory of Veterinary Public Health, College of Bioresource Sciences, Nihon University, Kanagawa, Japan. The antibody titers to B. henselae were determined by the indirect fluorescent antibody test (IFA), using B. henselae (ATCC 49882) as an antigen. The procedure followed a previous study by Maruyama et al. (2000). In brief, a volume of 10 μl of diluted serum (diluted in PBS containing 5% skim milk) was put onto test holes and slides were incubated at 37oC for 1 hour in a humid chamber. Then, the slides were washed twice with PBS for 15 min. Fluorescein-conjugated goat anti-cat immunoglobulin G (Cappel Products, Organon Teknika Corp, USA) was diluted 1:800 in PBS with 0.001% Evan’s blue and 10 μl of the mixture was applied into each well. Fluorescein-conjugated antidog immunoglobulin G was also employed in the IFA. The slides were incubated at 37oC for 1 hour, washed twice with PBS for 15 min, and then washed again with double distilled water for 10 min. The intensity of the bacillus-specific fluorescence was scored subjectively from +1 to +4, and the fluorescence score of ≥ +2 at a dilution of 1:32 was considered to be positive. Serum samples were screened at 1:32 and any positive sample at 1:32 dilution was titrated in a series of two fold dilutions up to 1:1,024. Statistical analysis: Pearson Chi-square tests were used to examine statistical significance; p<0.001 was considered significant.
Results and Discussion The cat samples collected from the 3 provinces range from 15-22 samples/province (an average of 18.7) (Table 1). All cat samples were mixed type. The number of antibody titers against B. hensalae in cats at the cut-off value 1:32, 1:64 and 1:128 were 5, 11, 5 samples, respectively (Table 2). The prevalence of antibodies to B. henselae varied by provinces, ranging from 0.0 to 73.0%. The average percentage of positive rate to B. henselae in Nan, Maehongson and Chiangrai provinces was 0.0, 52.0 and 73.0%, respectively with the cut-off value of 1:32 (Table 3). We found that the prevalence of antibodies to B. henselae in the 3 provinces had statistical differential (p<0.001). The prevalence of B. hensalae in Chiangrai and Maehongson provinces was significantly higher
than that of Nan province (p<0.001) but the prevalence of B. hensalae in Chiangrai and Maehongson provinces had no statistical differences. In this study, the gender and age of the cat had no statistical differences associated with seroprevalence. Examining the prevalence of antibodies to B. henselae in the cats in the two different border parts of Thailand, we found that the prevalence of antibodies to B. henselae from Thailand-Myanmar border (Chiangrai and Maehongson provinces, 61.8%) was significantly higher than that of Thailand-Laos PDR border (Nan province, 0.0%)(p<0.001). The dog samples collected from the 3 provinces range from 1560 samples/ province (an average of 38) (Table 1). All dogs sample were mixed type. This study found that the dogs in the 3 provinces along Thai-Myanmar-Laos PDR had no prevalence of antibodies against B. henselae. Table 2
Antibody titers against B. hensalae in cats along the northern borders of Thailand
Titer <1:32 1:32 1:64 1:128
Chiangrai 4 3 5 3
Province Maehongson 9 2 6 2
Total (56) 35 5 11 5
Nan 22 0 0 0
Table 3 The seroprevalence of B. hensalae IgG in cats along the northern borders of Thailand (cut off 1:32) Border Province Positive/ Samples % Positive
Thailand Myanmar Thailand Lao PDR Chiangrai Maehongson Nan Total 11/15
The seroprevalence of B. henselae in cats was found in Thailand-Myanmar Border (Chiangrai and Maehongson provinces), but not found in ThailandLoas PDR border (Nan province). The result correlated with report of isolate B. henselae from cats and cat fleas in Thailand-Myanmar Border (Parola et al., 2003). The seroprevalence in cats was higher in Chiangrai and Maehongson provinces, while no positive cats were found in Nan province. Chiangrai and Maehongson provinces are located in city site, but Nan province is located in rural and mountain areas. The seroprevalence of B. henselae in cats may depend upon the areas that the samples of sera were collected. The result also correlated with a report
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stating that warm and humid climates are strongly associated with the presence of antibodies against B. henselae and ectoparasite infestation in cats (Maruyama et al., 2001; Chomel et al., 2004). The antibody to B. henselae was found only in the cats and not found in the dogs captured from the 3 areas suggesting that cats are important reservoirs of B. henselae in these areas and host specificity may be responsible for the association of B. henselae with the feline reservoir (Breitschwerdt and Kordick, 2000). It is known that dogs serve as the reservoir of B. vinsonii subsp. berkhoffii. Recently, there was a report that dogs could be accidentally infected with B. henselae and B. clarridgeiae (Diniz et al., 2007; Inoue et al., 2009). Therefore, the other panels, B. vinsonii subsp. berkhoffii and B. clarridgeiae, should also be examined in the dog sera. In this study, we didn’t collect other kinds of samples e.g. mite, chigger, tick, flea, rat and humans to examine for B. hensalae by using isolation, purification, PCR, sequencing and phylogenetic classification techniques. Therefore, further studies should be conducted to clarify the epidemiology of the other reservoirs as the sources of Bartonell spp. infection along Thailand borders.
Acknowledgements We would like to gratefully acknowledge the fund for this project from the Regional Research Institute of Agricultural Production (RRIAP), College of Bioresource Sciences, Nihon University (NUBS). We also appreciated the knowledge from Drs. Toshikatsu Hagiwara, Kai Inoue, Chi Shih-Hui, Shingo Sato, Maskiet Boonyarith and Naris Tengchaisri. We would also like to thank the zoonosis team of National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand for kindly collecting the samples.
References Anderson, B.E. and Neuman, M.A. 1997. Bartonella spp. as emerging human pathogens. Clin
Microbiol Rev. 10: 203-219. Breitschwerdt, E.B. and Kordick, D.L. 2000. Bartonella infection in animals: Carriership, reservoir potential, pathogenicity, and zoonotic potential for human infection. Clin Microbiol Rev. 13: 428438. Chomel, B.B., Boulouis, H.J. and Breitschwerdt, E.B. 2004. Cat scratch disease and other zoonotic Bartonella infections. J Am Vet Med Assoc. 224: 1270-1279. Diniz, P.P.V.D.P., Maggi, R.G., Schwartz, D.S., Cadenas, M.B., Bradley, J.M., Hegarty, B. and Breitschwerdt, E.B. 2007. Canine bartonellosis: serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii. Vet Res. 38: 697-710. Inoue, K., Maruyama, S., Kabeya, H., Kawanami, K., Yanai, K., Jitchum, S. and Jittaparapong, S. 2009. Prevalence of Bartonella infection in cats and dogs in a metropolitan area, Thailand. Epidemiol Infect. 137(11): 1568-1573. Lamas, C.C., Curi, A., Bóia, M.N. and Lemos, E.R.S. 2008. Human bartonellosis: seroepidemiological and clinical aspects with emphasis on data from Brazil- A review. Mem Inst Oswaldo Cruz. 103: 221-235. Maruyama, S., Boonmar, S., Morita, Y., Sakai, T., Tanaka, S., Yamaguchi, F.F., Kabeya, H., and Katsube, Y. 2000. Seroprevalence of Bartonella henselae and Toxoplasma gondii among healthy individuals in Thailand. J Vet Med Sci. 62: 635637. Maruyama, S., Sakai, T., Morita, Y., Tanaka, S., Kabeya, H., Boonmar, S., Poapolathep, A., Chalarmchaikit, T., Chang, C., Kasten, R., Chomel, B. and Katsube, Y. 2001. Prevalence of Bartonella species and 16S rRNA gene types of Bartonella henselae from domestic cats in Thailand. Am J Trop Med Hyg. 65: 783-787. Parola, P., Sanogo, O.Y., Lerdthusnee, K., Zeaiter, Z., Chauvancy, G., Gonzalez, J.P., Miller, R.S., Telford. S.R., Wongsrichanalai, C. and Raoult, D. 2003. Identification of Rickettsia spp. and Bartonella spp. in from the Thai-Myanmar border. Ann N Y Acad Sci. 990: 173-181.
Presence of Infectious Pancreatic Necrosis Virus on Rainbow Trout (Oncorhynchus mykiss) by Histopathology, ELISA and RT-PCR Gabriel Aguirre-Guzmán* Ned Iván de la Cruz-Hernández Jesús Genaro Sánchez-Martínez
Abstract Trout (Oncorhynchus mykiss) culture in Mexico experiences sporadic and significant fish mortalities, where disease signs are associated with infectious pancreatic necrosis virus disease (IPNV). The purpose of the present work was to support the potential of ELISA, histopathology, and RT-PCR as routine techniques for IPNV detection. Fourteen trout farms were monitored after a disease outbreak. Positive results were confirmed in different collected organisms from 21% of tested farms by all 3 techniques. Virus detection by ELISA and RT-PCR was successfully performed in only two days after the initial signs of disease were observed in fish, since these methods are faster when compared to histopathology, which only detected signs of disease such as necrosis of the pancreatic acinus and intestine after 5 days. The results suggest that ELISA and RT-PCR offer an early, sensitive, faster and cheaper alternative for the routine detection of IPNV compared to cell culture and histopathology. These techniques can be performed for fish health monitoring and are reliable tools in the control, prevention and timely detection of IPNV. Keywords: ELISA, infectious pancreatic necrosis, trout, reverse transcription polymerase chain reaction 1 Facultad
de Medicina Veterinaria y Zootecnia. Universidad Autónoma de Tamaulipas. Km. 5 Carr. Cd. Victoria - Mante,
Thai J Vet Med. 2011. 41(1): 99-103.
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Introduction Infectious pancreatic necrosis virus (IPNV) of the Aquabirnavirus genus and Birnaviridae family is a well-known pathogen of rainbow trout (O. mykiss). This virus is one of the major etiological agents found in farms and wild salmonid fish, and its genome has a double-stranded RNA packaged in a nonenveloped icosahedral shell, 60 nm in diameter (Cutrín et al., 2000; Zhang and Suzuki, 2004). This is a highly contagious disease initially detected in North America (Roberts and Pearson, 2005) with mortalities ranging from 70-100% in fingerlings and early juvenile fish, usually following culture stress (Evensen and Lorenzen, 1997). The susceptibility to this virus decreases with age, but asymptomatic organisms facilitate horizontal and vertical transmission (Brown and Bruno, 2002). Clinical signs include skin hyperpigmentation, distended abdomen with ascites, empty gut, presence of clear or milky mucus in stomach and anterior intestine, long, thin white fecal casts, spiral swimming, pale spleen, heart, liver, and petechiae observed in the viscera (Crane et al., 2000; Brown and Bruno, 2002).
Rainbow trout farming in Mexico is a growing and new industry whose production increased from 1612 to 11,792 metric tons in one decade (1998-2008); with most of the rainbow trout farms and hatcheries located in Chihuahua, Durango, Mexico, Michoacán, Morelos, and Puebla (CONAPESCA 2008). However, other states such as Hidalgo, Jalisco, Nuevo León, and San Luis Potosi have small scale extensive farms with potential to increase their production because of their association to eco-tourism and proximity to big cities. The first report of IPNV in Mexico occurred in 2002, from O. mykiss samples obtained from a farm located in Central Mexico (Ortega et al., 2002); however, IPNV distribution in farm and wild stock in Mexico has been little understood and poorly evaluated (BarreraMejia et al., 2002). The standard protocol for the routine detection of IPNV is usually based on virus isolation (Milne et al., 2006), complemented by histopathology, immunofluorescence, serology, and molecular biology (Barrera-Mejia et al., 2002; Bowden et al., 2002; Jenĉiĉ et al., 2002). Viral isolation is considered as the gold standard, but it is laborious, expensive and time consuming (Kerr and Cunningham, 2006; Milne et al.,
Aguirre-Guzmán G. et al. / Thai J Vet Med. 2011. 41(1): 99-103. 2006). The purpose of the present study was to evaluate the use of RT-PCR and ELISA for the routine diagnosis of IPNV in aquatic health labs.
Materials and Methods Fourteen rainbow trout farms in the Mexican state of Hidalgo (Fig. 1) were monitored for the presence of IPNV virus. Four hundred and ninety six fish (O. mykiss) showing clinical signs such as erratic swimming, loss of appetite, skin darkening, distended abdomen, exophthalmia, and long thin white fecal casts were collected. Fingerlings and early juveniles were collected without dissection and immediately submerged into 10% neutral buffered formalin (Evensen and Lorenzen 1997). Older juveniles were dissected in situ and their gills, brain, heart, spleen, kidney, liver, pancreas, and intestine samples were collected (Barlic-Maganja et al., 2002; Kerr and Cunningham, 2006). The ELISA samples were washed with sterile PBS and stored at -80ºC until used. Samples for RT-PCR were washed with PBS and placed in RNAlater® (Ambion, USA) for storage (Phelan et al., 2005). The fixed samples (whole organisms or tissues) were processed for routine histology; embedded in paraffin according to standard procedures, sectioned at 4-6 µm, stained with H&E. The samples were examined by light microscopy (Carl Zeiss/Axiostar) coupled to a digital camera (Canon, Powershot G6 PC1089) to look for IPNV-associated morphologic changes such as necrotic lesions and ulcers in the pancreatic acini, esophagus, stomach, intestine, and renal hematopoietic elements (Bowden et al., 2002; Roberts and Pearson, 2005). Fish samples from the same pond were pooled in groups of three or four organs, and homogenized with a pellet mixer (VWR, USA) at 200 rpm for 1 min with sterile PBS, and independently analyzed. All samples were tested with an ELISA kit for IPNV (Test-line®, Czech Republic) according to
Figure 1 Trout (Oncorhynchus mykiss) farm locations in Hidalgo state, Mexico, and yearly aquaculture yield.
the manufacturer’s instructions (Bowden et al., 2002; Jenĉiĉ et al., 2002). The samples were analyzed with a Bio-Rad (USA) microplate reader at 415 nm, and were positive when absorbance was greater than 0.5. Positive controls used were supplied by Laboratorio de Biotecnología 1, Facultad de Ciencias Químicas, Universidad Autonoma de Chihuahua (LB1-FCQUACH), and the test-line kit. Rainbow trout samples (whole organisms or tissues) were sent for RT-PCR analysis to the LB1FCQ-UACH. All samples were pooled in groups of three or four organs, homogenized with a pellet mixer at 200 rpm for 1 min with sterile RNAlater® at 4ºC. All samples were processed with a RNA isolation kit (Quantum Prep AquaPure Genomic) according to the manufacturer’s instructions (Holmes et al., 2003). Reverse transcription reactions were performed using the protocol of SuperScriptTM II ARNse reverse transcriptase (Invitrogen Inc., USA)(Cutrín et al., 2000). For IPNV detection, PNF primers [ATCTGCGGTGTAGACATCAAA (Forward) and TGCAGTTCCTCGTCCATCCC (Reverse)](Taksdal et al., 2001; Barrera-Mejia et al., 2002) were used for the PCR reaction where the visualization of a 224 bp band was considered as a positive result (Fig. 2). PCR was carried out in a total volume of 25 µl of FSB buffer (10 mM dNTP’s, 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, pH 8.3) containing 15 pmol of each primer, and 2.5 U of Taq polymerase (Promega, Inc.). The PCR reactions were carried out in a thermocycler (Corbett Research) under the following conditions: 96ºC for 5 min followed by 94ºC for 60 sec (denaturalization), 55ºC for 30 sec (annealing) and 72ºC for 60 sec (extension). After 35 cycles, the reactions were cooled down to 4ºC, and PCR products were analyzed on 2% agarose gel in 1x TBE buffer (89 mM Tris HCl, 89 mM boric acid, 2 mM EDTA, pH 8) at 80 V for 60 min. The gels were stained with ethidium bromide, and analyzed with imaging system software from Kodak (Model Gel logic-200).
Figure 2 IPNV amplification from trout samples in 2% agarose gel electrophoresis in 1x TBE at 80 volts by 60 min. MWM: molecular weight marker; Lane 1 and 2: negative control (trout tissue sample and distillate sterile water, respectively); Lane 3: positive control from LB1-FCQ-UACH; Lane 4-6: trout samples.
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Figure 3 Histological sections from Oncorhynchus mykiss. (A) Acute necrosis in pancreas (arrow) (H&E, 4x). (B) Generalized necrosis in intestine (arrow) and cells with nuclear pyknosis and karyorrhexis (arrow head)(H&E, 40x).
Results and Discussion The detection protocols for different pathogens (virus, bacteria, fungi, etc.) on aquatic organisms help to increase the knowledge about their presence, biogeographical distribution, effects, and control on aquaculture and fisheries production settings (Ghittino et al., 2003). In Mexico, rainbow trout production areas have shown a sudden and significant increase in mortality in fingerlings and early-juveniles during 2003-2006 (CONAPESCA 2008), which had a direct effect on fish farms. The international diagnosis protocol for IPNV is based on virus isolation by cell culture; which is a laborious, expensive and time-consuming activity. Histology, immunofluorescence, immunohistochemistry (Ellis et al. 2010), ELISA (Bowden et al., 2002; Jenĉiĉ et al., 2002), and RT-PCR (Taksdal et al., 2001; Barrera-Mejia et al., 2002) are other methods that have been suggested as complementary for IPNV detection, because of their sensitivity, speed and economy. In this study, the organisms collected (496) from rainbow trout farms (14) consisted of fingerlings (13%), early-juveniles (26%), juveniles (52%) and adults (9%). Significant histopathological lesions were observed in the different fish samples examined; however, only samples from farms 1, 4 and 8 showed lesions associated to IPNV, such as pancreatic necrosis, necrotizing enteritis; mucosal epithelial necrosis and catharrhal enteritis (Fig 3). Renal tissues displayed moderate degenerative tubules with necrotic areas (Brown and Bruno, 2002; Roberts and Pearson, 2005). However, the usefulness of histopathology in IPNV diagnosis is only effective when: i) a specific sign is associated to a specific pathogen; ii) this specific sign is expressed at a high enough level; and iii) the aquatic pathologist experience. The histopathological changes observed in the collected samples were not exclusive of IPNV diseases and the use of other diagnostic protocols were necessary for a confirmed diagnosis of the specific pathogen affecting the organism. Table 1 shows the results of the ELISA and RT-PCR analyses for all trout samples. It can be seen that samples from three farms (1, 4, and 8; = 21% of
tested farms) were positive to IPNV (100%, pool of samples) by ELISA (1.12, 1.17, and 1.13 of S/P ratio mean, respectively) and RT-PCR, in a much shorter time frame compared to histopathology and exhibited a higher sensitivity to IPNV detection. Prevention of pathogen exposure is the most effective way of controlling disease, with farms including routine diagnostic examinations for their aquatic health programs. These techniques may represent an advantage for the routine and timely diagnosis of IPNV in trout culture and wild stock studies, since early pathogen identification is crucial in the prevention of spread of infection, and in decision making for treatment and disease control (Barrera-Mejia et al., 2002), which represent possible effective containment and elimination of an emergency disease outbreak and epidemic. Table 1 ELISA and RT-PCR results from trout farm of Hidalgo state, Mexico. Trout farm 1 2 3 4 5 6 7 8 9 10 11 12 13 14
*Reactive (R) where the mean absorbance in wells with positive antigen is < 0.5. ND: not determined. Control positive: T+ (sample from LB1-FCQ-UACH), K+ (control positive from test-line kit) Control negative: W- (distilled water).
Acknowledgements This study was funded by grants from the Mexican Council of Science and Technology (CONACyT), project SAGARPA-CONACYT-2003002-227). We thank Laboratorio de Biotecnología 1, Fac. de Ciencias Químicas, Universidad Autonoma de
Aguirre-Guzmán G. et al. / Thai J Vet Med. 2011. 41(1): 99-103. Chihuahua (Dr. G. Erosa de la Vega) for supplying the positive control and the laboratory facilities for this work.
References Barlic-Maganja, D., Strancar, M., Hostnik, P., Jencic, V. and Grom, J. 2002. Comparison of the efficiency and sensitivity of virus isolation and molecular methods for routine diagnosis of infectious haematopoietic necrosis virus and infectious pancreatic necrosis virus. J Fish Dis. 25: 73-80. Barrera-Mejia, M., Simon-Martinez, J., SalgadoMiranda, C., Vega, F., Ortega, C. and Aragon, A. 2002. Development and validation of a shorttime cell culture and multiplex reverse transcriptase polymerase chain reaction assay for infectious pancreatic necrosis virus in mexican farm-sampled rainbow trout. J Aquat Anim Health. 21: 167-172. Bowden, T.J., Smail, D.A. and Ellis, A.E. 2002. Development of a reproducible infectious pancreatic necrosis virus challenge model for Atlantic salmon, Salmo salar L. J Fish Dis. 25: 555563. Brown, L.L. and Bruno, D.W. 2002. Infectious diseases of coldwater fish in fresh water. In: Diseases and Disorders of Finfish in Cage Culture, P.T.K. Woo, D.W. Bruno and L.H.S. Lim (eds) CABI publishing, New York, 107-171. CONAPESCA (Comisión Nacional de Acuacultura y Pesca). 2008. Anuario Estadístico de Pesca. Crane, M.S.J., Hardy-Smith, P., Williams, L.W., Hyatt, A.D., Eaton, L.M., Gould, A., Handlinger, J., Kattenbelt, J. and Gudkovs, N. 2000. First isolation of an aquatic birnavirus from farmed and wild fish species in Australia. Dis Aquat Org. 43: 1-14 Cutrín, J.M., Barja, J.L., Nicholson, B.L., Bandin, I., Blake, S. and Dopazo, C.P. 2000. Restriction fragment length polymorphisms and sequence analysis: an approach for genotyping infectious pancreatic necrosis virus reference strains and other aquabirnaviruses isolated from northwestern Spain. Appl Environ Microbiol. 70: 1059-1067. Evensen, O. and Lorenzen, E. 1997. Simultaneous demonstration of infectious pancreatic necrosis virus (IPNV) and Flavobacterium psychrophilum in paraffin embedded specimens of rainbow trout Oncorhynchus mykiss fry by use of paired immunohistochemistry. Dis Aquat
Org. 29: 227-232. Ghittino, C., Latini, M., Agnetti, F., Panzieri, C., Lauro, L., Ciappelloni, R. and Petracca, G. 2003. Emerging pathologies in aquaculture: Effects on production and food safety. Vet Res Comm. 27: 471-479. Ellis, A.E., Cavaco, A., Petrie, A., Lockhart, K., Snow, M. and Collet, B. 2010. Histology, immunocytochemistry and qRT-PCR analysis of Atlantic salmon, Salmo salar L., post-smolts following infection with infectious pancreatic necrosis virus (IPNV). J Fish Dis. 33: 787-863. Holmes, S.P., Witbaard, R. and van der Meer, J. 2003. Phenotypic and genotypic population differentiation in the bivalve mollusc Arctica islandica: Results from RAPD analysis. Mar Ecol Prog Ser. 254: 163-176. Jenĉiĉ, V., Hostnik, P., Barliĉ Maganja, D. and Grom, J. 2002. The spread of salmonid viral diseases in Slovenia. Slov Vet Res. 39: 197-205. Kerr, C. and Cunningham, C. 2006. Moving molecular diagnostics from laboratory to clinical application: A case study using infectious pancreatic necrosis virus serotype A. Lett Appl Microbiol. 43: 98-104. Milne, S.A., Gallacher, S., Cash, P. and Porter, A.J.R. 2006. A reliable RT-PCR–ELISA method for the detection of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout. J Virol Methods. 132: 92-96. Ortega, C., Montes de Oca, R., Groman, D., Yason, C., Nicholson, B. and Blake, S. 2002. Viral infectious pancreatic necrosis in farmed rainbow trout from Mexico. J Aquat Anim Health. 14: 305-310. Phelan, P.E., Pressley, M.E., Witten, P.E., Mellon, M.T., Blake, S. and Kim, C.H. 2005. Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio). J Virol. 79: 1842-1852. Roberts, R.J. and Pearson, M.D. 2005. Infectious pancreatic necrosis in Atlantic salmon, Salmo salar L. J Fish Dis. 28: 383-390. Taksdal, T., Dannevig, B.H. and Rimstad, E. 2001. Detection of infectious pancreatic necrosis (IPN) virus in experimentally infected atlantic salmon parr by RT-PCR and cell culture isolation. Fish Pathol. 21: 214-218. Zhang, C.X. and Suzuki, S. 2004. Aquabirnaviruses isolated from marine organisms form a distinct genogroup from other aquabirnaviruses. J Fish Dis. 27: 633-643.
The Investigation of the Relations between Insulin-liked Growth Factor-I and Body Weight and between Insulin-liked Growth Factor-I and Sex in Young Cats Wei-Yau Shia1 Anchana Songkaew1 Sasisopa Singhanetr1 Chih-Chung Chou1, 2 Wei-Ming Lee1, 2*
Abstract High plasma IGF-I concentration has been found in large breed young dogs, but not in young cats. The objective of this study was to investigate the relation between the concentration of IGF-I and body weight in both male and female cats at young age. In this study, the plasma IGF-I concentrations and body weight in 4 female and 4 male cats at 5, 11, 17 and 21 months old were examined. No significant difference in the body weight between the female and male kittens (3.1±0.2 kg and 3.2±0.3 kg) at 5 months old was found, but at 11, 17 and 21 months old (5.3±0.2 kg, 5.3±0.4 kg, 5.4±0.2 kg) the male cats had significantly higher body weight (p<0.01) than the female cats (3.1± 0.3 kg, 3.3± 0.2 kg, 3.4±0.3 kg). The IGF-1 concentration in the male cats (945±41 ng/ml) was significantly higher (p<0.05) than that in the female cats (520±39 ng/ml) at the age of 5 months. At 11 and,17 months old, but not at 21 months old, the mean plasma IGF-1 levels (772±122 and 713±33 ng/ml) in the male cats were significantly higher than those in the female cats (323±77 and 197±36 ng/ml) (p<0.05). In conclusion, the results of this study indicated that there were relations between IGF-I and body weight and between IGF-I and sex in young cats. Moreover, the diversity of plasma IGF-1 concentrations in male and female cats can be found at the age of 5 months and over. Keywords: body weight, cats, IGF-I, sex 1 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung City, 402 Taiwan, R.O.C. 2Veterinary Medical Teaching Hospital, College of Veterinary Medicine, National Chung Hsing University, 250-1, Kuo Kuang Rd., Taichung City, 402 Taiwan, R.O.C. Corresponding author E-mail: [email protected]
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Shia W. et al. / Thai J Vet Med. 2011. 41(1): 105-109.
บทคัดย่อ การศึกษาความสัมพันธ์ระหว่าง Insulin-liked Growth Factor-I และน้ําหนักตัวและเพศในลูก แมว Wei-Yau Shia1 Anchana Songkaew1 Sasisopa Singhanetr1 Chih-Chung Chou1, 2 Wei-Ming Lee1, 2* ค่าความเข้มข้นพลาสมา IGF-I มีรายงานการตรวจพบในระดับสูงในลูกสุนัขพันธุ์ใหญ่ แต่ไม่พบในลูกแมว วัตถุประสงค์ของ การศึกษาในครั้งนี้ เพื่อหาความสัมพันธ์ระหว่างค่าความเข้มข้นพลาสมา IGF-I และน้ําหนักตัวในลูกแมวทั้ง 2 เพศ โดยทําการตรวจค่าความ เข้มข้นพลาสมา IGF-I ในแมวเพศเมียและเพศผู้ จํานวนเพศละ 4 ตัว ที่ช่วงอายุ 5 11 17 และ 21 เดือน ตามลําดับ ผลการศึกษาพบว่า น้ําหนักตัวของลูกแมวเพศผู้และเมีย มีค่าเท่ากับ 3.1±0.2 กก. และ 3.2±0.3 กก. ที่ช่วงอายุ 5 เดือน ซึ่งไม่มีความแตกต่างอย่างมีนัยสําคัญ ในขณะที่แมวเพศผู้ช่วงอายุ 11 17 และ 21 เดือนมีค่าน้ําหนักตัวเท่ากับ 5.3±0.2 กก. 5.3±0.4 กก. และ 5.4±0.2 กก. ซึ่งพบว่าค่าดังกล่าว สูงกว่าแมวเพศเมีย ซึ่งมีค่าเท่ากับ 3.1±0.3 กก. 3.3±0.2 กก. และ 3.4±0.3 กก. อย่างมีนัยสําคัญทางสถิติ(p<0.01) ในช่วงอายุเดียวกัน ค่า ความเข้มข้นพลาสมา IGF-I ในแมวเพศผู้ (945±41 ng/ml) มีค่าสูงแตกต่างอย่างมีนัยสําคัญ (p<0.05) กว่าแมวเพศเมีย (520±39 ng/ml) ที่ ช่วงอายุ 5 เดือน ที่ช่วงอายุ 11 และ17 เดือน ยกเว้น 21 เดือน ค่าเฉลี่ยความเข้มข้นพลาสมา IGF-I ในแมวเพศผู้ มีค่า (772±122 และ 713±33 ng/ml) ซึ่งมีค่าสูงกว่าแมวเพศเมีย อย่างมีนัยสําคัญทางสถิติ (323±77 และ 197±36 ng/ml) (p<0.05) สรุปผลการศึกษาพบว่า มี ความสัมพันธ์ระหว่างค่าความเข้มข้นพลาสมา IGF-I และน้ําหนักตัว และเพศในลูกแมว ซึ่งแสดงผลอย่างชัดเจนในช่วงแมวอายุมากกว่า 5 เดือนขึ้นไป คําสําคัญ: น้ําหนักตัว แมว IGF-I เพศ 1 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung City, 402 Taiwan, R.O.C. 2 Veterinary Medical Teaching Hospital, College of Veterinary Medicine, National Chung Hsing University, 250-1, Kuo Kuang Rd., Taichung City, 402 Taiwan, R.O.C. *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction The plasma concentration of insulin-like growth factor-1 (IGF-1) is regulated by growth hormone (GH), which stimulates IGF-1 synthesis in the liver and other tissues. IGF-1 participates in the growth and function of almost every organ in the body (Müller et al., 1999; Kitiyanant et al., 2003). There is a complex interplay in the metabolic actions of GH and IGF-1. In circulation, IGF-1 is transported in the serum bound to specific high-affinity binding proteins (IGFBP-1 to IGFBP-6) mainly produced in the liver. Most of the circulating IGF-1 (more than 90– 95%) is bound to IGF-binding protein-3 (IGFBP-3) in a ternary complex with an acid-labile subunit (ALS) (Lin-Su and Wajnrajch, 2002; Kitiyanant et al., 2003; Mak et al., 2008). Some studies have been documented that tissues that determine the regulation of GH and IGF-1 peptide and receptors may be controlled by sex steroid. For example, in rats, estradiol can induce the synthesis of IGF-1 and its receptor and alter the availability of selected IGFBPs in the central nervous
system (CNS), pituitary gland, ovary, breast, uterus, bone, and pre-adipocytes while inhibiting IGF-1 production by the liver. Estradiol can also stimulate the hepatic (subtype 1) GH receptor, but represses in the hypothalamus. Testosterone and nonaromatizable androgens induce IGF-1 peptide in bone, muscle, and skin, but suppress type I IGF receptor in fat (Veldhuis et al., 2006). Besides the regulation of IGF-1 by the sex steroids, the concentrations of IGF-1 might also be an important maturation process in animals. In fact, puberty is an endocrinologically transitional process that marks progression from childhood to adulthood. The concurrent activation of the somatotropic and gonadotropic axes is a hallmark of this physiological transition (Veldhuis et al., 1997). In human, it has been reported that IGF-1 concentration increases with age in prepubertal boys (Guercio et al., 2002) and girls (Guercio et al., 2003). In fact, in dogs body size is associated with the level of IGF-I (Eigenmann et al., 1988). For dogs, their body sizes are associated with the level of IGF-I (Eigenmann et al., 1988). Therefore, large breed dogs present a higher concentration of
Shia W. et al. / Thai J Vet Med. 2011. 41(1): 105-109. IGF-I in comparison with small breed dog at a young age (Favier et al., 2001). As for cats, IGF-I concentrations were detected in cats ranging from four to sixteen years old, but no significant differences in the concentration between the sexes were shown (Reusch et al., 2006). Moreover, information concerning the differences in the concentration of IGF1 between male and female cats less than two years old is limited. Thus, the aim of this study was to investigate the concentration of IGF-I and its relation to body weight in both male and female cats at a young age.
Materials and Methods Animals: Eight healthy mixed breed cats including 4 males and 4 females were used in this study. The cats were purchased at 2 months of age from a cat shelter in Taichung, Taiwan. They were raised together in a bright (12 hours per day) and temperature (25-28oC) controlled room. The cats were fed with standard commercial dry food and provide with water ad libitum. The cats used in this study were 5, 11, 17 and 21 months old. All blood drawing was carried out in conscious animals after overnight fast. The study was approved by Affidavit of Approval of Animal use Protocol, National Chung Hsing University. Blood sampling for IGF-1 measurement: The blood samples for IGF-1 were collected from the cephalic vein of cats 5, 11, 17 and 21 months old. The collected blood samples were immediately transferred to EDTA-coated tubes, and chilled at 4oC. Plasma was separated by centrifuge at 1000 x g for 10 minutes, thereafter they were transferred to tubes and stored at -70oC until IGF-1 determination. Plasma IGF-1 was measured by two-site radioimmunometric assay (IRMA) (ACTIVE® Non-Extraction IGF-1 IRMA DSL2800). The minimum concentration of IGF-1 that could be detected was 2.06 ng/ml. The intra- and inter coefficients of variance (CVs) for GH assay were 7.0 and 7.4%, respectively. Statistical analysis: Values are presented as mean ± standard error of mean (SEM) and statistically analyzed using the SigmaPlot software version 9.0. The change of plasma hormone levels was tested by Analysis of variance (one-way ANOVA) using SAS version 9.1 software (SAS institute, Inc., Cary, NC). The significance of the difference between two means was tested using Mann-Whitney U test (SigmaPlot® 9.0). A p value of <0.05 was considered to be significant.
107 Table 1 Body weight (kg) of cats at different ages.
Female Mean±SEM Male Mean±SEM
3.7 2.9 2.8 2.8 3.1±0.2 3.9 2.8 2.8 3.3 3.2±0.3
3.7 2.9 3.4 2.4 3.1±0.3 5.2 5.8 5.0 5.0 5.3±0.2
3.9 3.2 3.2 2.7 3.3±0.2 5.1 6.3 4.6 5.0 5.3±0.4
4.2 3.3 3.3 2.6 3.4±0.3 5.2 5.9 5.1 5.3 5.4±0.2
Results and Discussion The individual and average body weights of the cats age 5, 11, 17 and 21months old are shown in Table 1. There was no significant difference in body weight between the female and male kittens (3.1±0.2 kg and 3.2±0.3 kg) at 5 months old (Fig. 1). The body weight of the male cats at the age of 11, 17 and 21 months old (5.3±0.2 kg, 5.3±0.4 kg, 5.4±0.2 kg) was significantly higher (p<0.05) than that of the female cats (3.1±0.3 kg, 3.3±0.2 kg, 3.4±0.3 kg) (Fig. 1). The mean plasma concentrations of IGF-1 in cats at the age of 5, 11, 17 and 21 months are shown in Table 2. The mean plasma IGF-1 concentrations in the male cats were significantly higher than those in the female cats at 5, 11 and 17 months old (Fig. 2). In male cats, the mean plasma IGF-1 at 5 months old (945±41 ng/ml) was significantly higher (p<0.05) than at 17 and 21 months old (713±33 and 648±79 ng/ml) (Fig. 3). In female cats, the basal plasma IGF-1 level at 5 months old (520±39 ng/ml) was significantly higher (p<0.05) than that at 17 months old (197±36 ng/ml), but did not differ significantly at the age of 11
Figure 1 The body weight (kg) of the male and female cats at the age of 5 months, 11 months, 17 months and 21 months * indicates significant difference between male and female cats at the same age.
Shia W. et al. / Thai J Vet Med. 2011. 41(1): 105-109. Table 2 Basal plasma IGF-1 concentration (ng/ml) of cat at different ages.
Figure 2 The concentrations (ng/ml) of IGF-1 in the male and female cats at the age of 5 months, 11 months, 17 months and 21 months. * indicates significant difference between the male and female cats of the same age.
and 21 months (323±77 and 518±79 ng/ml). The highest mean plasma IGF-1 concentrations were found at the age of 5 months after which it decreased with age. This might be associated with the lack of gonadal hormones. In fact, the first heat in queens can be as early as 4 or as late as 21 months depending on the breed (Jemmett and Evans, 1977). The concentrations of IGF-I can decrease by the effect of gonadal hormone. For example, IGF-1 concentrations increase significantly after castration in male cats (Martin et al., 2006), while oestrogen can attenuate GH action by reducing IGF-1 production (Mauras et al., 2006). However, the concentration of IGF-I increases as age increases in prepubertal and pubertal boys and girls (Guercio et al., 2002; Guercio et al., 2003). It is suggested that IGF-1 is able to stimulate proliferation in Leydig cells (Colón et al., 2007) and maturation of oocytes (Kitiyanant et al., 2003). Thus, the elevated concentrations of IGF-I observed at the age of 5 month old in kittens in this study might be an important maturation process for puberty in cats. The mean concentrations of IGF-1 of 21 month-old female cats (518±79 ng/ml) was significantly higher (p<0.05) than that of 17 month-old female cats (197±36 ng/ml) (Fig. 4). The mean plasma IGF-1 level in the male cats was significantly higher than in the female cats at almost all ages in this study, except for the 21-month old group. This may be ascribed to the effect of sex steroids. After puberty, sex steroids can modulate IGF-1 secretion. In male cats, testosterone mediates GH action at the hypothalamic level by GHRH increased GH secretion with acromegalic increase in IGF-I level. In female cats, oestrogen attenuates GH action by reducing IGF1 (Meinhardt and Ho, 2006). Furthermore, the production of IGF-1 may be associated with the secretory patterns of GH. In fact, the different GH secretory patterns have been reported in different genders (Veldhuis et al., 2000). Therefore, the higher IGF-1 concentration noted in 5 month old cats might be due to the high GH secretory burst mass. The elevated IGF-1 concentration in the male cats compared to female cats at a young age may indicate a different sensitivity of IGF-1 response to GH. Indeed, in humans, males experience a higher
Figure 3 The mean concentrations of plasma IGF-1 in the male cats at the age of 5 months, 11 months, 17 months and 21 months. * indicates significant difference at 17 and 21 months old compared to 5 months old.
Figure 4 The mean concentrations of plasma IGF-1 in the female cats at the age of 5 months, 11 months, 17 months and 21 months. * indicates significant difference at 17 months old compared to 5 months old; # indicates significant difference at 17 months old compared to 21 months old.
response of IGF-1 to GH treatment (Mauras et al., 2007). Taken together; it may explain the result of this study, which showed higher IGF-1 concentrations in 5 month-old cats and a decrease in older age. To our knowledge, the gender diversity of plasma IGF-1 from 5 months old in cats has not been previously reported. At 21 months old, the basal plasma IGF-1 level of the female cats was elevated when compared with that at 11 months old. It has been reported that elevated plasma IGF-1 was observed 2 days before behavioral estrous in goats (Hashizume et al., 2000). Thus, the elevated IGF-1 concentration at the age of 21 months in this study may be related to the effect of
Shia W. et al. / Thai J Vet Med. 2011. 41(1): 105-109.
sex steroids. Actually, the effect of oestrogen on the secretion of IGF-1 is still controversial. For example, in humans, high oestrogen concentration in the portal circulation impairs hepatic IGF-1 (Lin-Su and Wajnrajch, 2002; Moyano and Rotwein, 2004; Reusch et al., 2006), but in rats, oestradiol induces synthesis of IGF-1 and its receptors in such a way that it alters the availability of selected IGFBPs in the central nervous system (CNS), pituitary gland, ovary, breast, uterus, bone, and preadipocytes while inhibiting IGF-1 production by the liver (Veldhuis et al., 2006). IGF-1 is localized in the endometrial stroma and underlying myometrium, and its expression at both the mRNA and protein levels is shown to increase during the proliferative phase of menstrual cycle and in response to oestrogen in experiment animals. Several evidences have indicated that oestrogen is a key stimulator of IGF-1 gene and protein expression in the uterus (Moyano and Rotwein, 2004).
in goats. Domest Anim Endocrinol. 18(2): 253263. Jemmett, J.E. and Evans, J.M. 1977. A survey of sexual behaviour and reproduction of female cats. J Small Anim Pract. 18(1): 31-37. Kitiyanant, Y., Saikhun, J. and Pavasuthipaisit, K. 2003. Somatic cell nuclear transfer in domestic cat oocytes treated with IGF-1 for in vitro maturation. Theriogenology 59(8): 1775-1786. Leung, K.C., Johannsson, G., Leong, G.M. and Ho, K.K. 2004. Estrogen regulation of growth hormone action. Endocr Rev. 25(5): 693-721. Lin-Su, K. and Wajnrajch, M.P. 2002. Growth hormone releasing hormone (GHRH) and the GHRH receptor. Rev Endocr Metab Disord. 3(4): 313-323. Mak, R.H., Cheung, W.W. and Roberts, C.T. 2008. The growth hormone–insulin-like growth factor-I axis in chronic kidney disease. Growth Horm IGF Res. 18(1): 17-25. Martin, L.J., Siliart, B., Dumon, H.J. and Nguyen, P. 2006. Spontaneous hormonal variations in male cats following gonadectomy. J Feline Med Surg. 8(5): 309-314. Mauras, N., Bishop, K. and Welch, S. 2007. Growth hormone action in puberty: effects by gender. Growth Horm IGF Res. 17(6): 463-471. Meinhardt, U.J. and Ho, K.K. 2006. Modulation of growth hormone action by sex steroids. Clin Endocrinol (Oxf). 65(4): 413-422. Moyano, P. and Rotwein, P. 2004. Mini-review: estrogen action in the uterus and insulin-like growth factor-I. Growth Horm IGF Res 14(6): 431-435. Müller, E.E., Locatelli, V. and Cocchi, D. 1999. Neuroendocrine control of growth hormone secretion. Physiol Rev. 79(2): 511-607. Perry, R.J., Farquharson, C. and Ahmed, S.F. 2008. The role of sex steroids in controlling pubertal growth. Clin Endocrinol (Oxf). 68(1): 4-15. Reusch, C.E., Kley, S., Casella, M., Nelson, R.W., Mol, J. and Zapf, J. 2006. Measurements of growth hormone and insulin-like growth factor 1 in cats with diabetes mellitus. Vet Rec. 158(6): 195-200. Veldhuis, J.D., Metzger, D.L., Martha, P.M. Jr., Mauras, N., Kerrigan, J.R., Keenan, B., Rogol, A.D. and Pincus, S.M. 1997. Estrogen and testosterone, but not a nonaromatizable androgen, direct network integration of the hypothalamo-somatotrope (growth hormone)insulin- like growth factor I axis in the human: evidence from pubertal pathophysiology and sex-steroid hormone replacement. J Clin Endocrinol Metab. 82(10): 3414-3420. Veldhuis, J.D., Roemmich, J.N. and Rogol, A.D. 2000. Gender and sexual maturation-dependent contrasts in the neuroregulation of growth hormone secretion in prepubertal and late adolescent males and females--a general clinical research center-based study. J Clin Endocrinol. Metab. 85(7): 2385-2394.
In conclusion, the difference of basal plasma IGF-1 level between sexes could be observed in cats since the age of 5 months, and male cats had significantly higher mean plasma IGF-1 levels than female cats. Thus, the relations between IGF-I and body weight and between IGF-I and sex in young cats and the differences in plasma IGF-I levels can be seen in the male and female cats at the age of 5 months and over.
Acknowledgements The authors would like to thank the help of English writing in this article by Professor Dr. Wong, Min-Liang and Dr. Amelia M.M. Jack.
References Colón, E., Zaman, F., Axelson, M., Larsson, O., Carlsson-Skwirut, C., Svechnikov, K.V. and Söder, O. 2007. Insulin-like growth factor-I is an important antiapoptotic factor for rat Leydig cells during postnatal development. Endocrinology. 148(1): 128-139. Eigenmann, J.E., Amador, A. and Patterson, D.F. 1988. Insulin-like growth factor I levels in proportionate dogs, chondrodystrophic dogs and in giant dogs. Acta Endocrinol(Copenh). 118(1): 105-108. Favier, R.P., Mol, J.A., Kooistra, H.S. and Rijnberk, A. 2001. Large body size in the dog is associated with transient GH excess at a young age. J Endocrinol. 170(2): 479-484. Guercio, G., Rivarola, M.A., Chaler, E., Maceiras, M. and Belgorosky, A. 2002. Relationship between the GH/IGF-1 axis, insulin sensitivity, and adrenal androgens in normal prepubertal and pubertal boys. J Clin Endocrinol Metab. 87(3): 1162-1169. Guercio, G., Rivarola, M.A., Chaler, E., Maceiras, M. and Belgorosky, A. 2003. Relationship between the growth hormone/insulin-like growth factorI axis, insulin sensitivity, and adrenal androgens in normal prepubertal and pubertal girls. J Clin Endocrinol Metab. 88(3): 1389-1393. Hashizume, T., Ohtsuki, K. and Matsumoto, N. 2000. Plasma insulin-like growth factor-I concentrations increase during the estrous phase
Veldhuis, J.D., Roemmich, J.N., Richmond, E.J. and Bowers, C.Y. 2006. Somatotropic and gonadotropic axes linkages in infancy, childhood, and the puberty-adult transition. Endocr Rev. 27(2): 101-140.
The Activity of Plasma Matrix Metalloproteinase-9 as a Marker to Reflect the Tissue Level of Mammary Glands Tumor in Dogs Wei-Yau Shia1 Shih-Ming Liu1 Chun-Sheng Lee2 Jian-Liang Lin2 Chih-Chung Chou1 Yumi Yuasa1 Shih-Chieh Chang1,2 Wei-Ming Lee1, 2*
Abstract In order to understand the change of MMP-2 and MMP-9 in dogs with mammary gland tumor (MGT), the activity of MMP-2 and MMP-9 in plasma samples, MGT tissues and near by normal mammary gland tissues were evaluated by gelatin zymography. The levels of MMP-2 and MMP-9 were significantly higher in tumor tissues than those in normal gland tissues (p<0.05). Plasma MMP-9 levels in malignant cases were higher than those in benign (p=0.15) and non-tumor cases (p<0.05). Likewise, plasma MMP-2 levels in malignant MGT cases were higher than non-tumor (p<0.01) and benign cases (p=0.54). Furthermore, plasma MMP-9 levels were reflected and positively correlated with MMP-9 in tumor tissues (r=0.78, p=0.02), but not MMP-2. Our results indicated that the MMP-2 and MMP-9 levels are associated with tumor malignancy, and plasma MMP-9 activity can become a potential marker both in follow-up and prognosis of canine MGT patients. Keywords: dog, mammary tumor, matrix metalloproteinase 1 Department
of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung City, 402 Taiwan, R.O.C. 2Veterinary Medical Teaching Hospital, College of Veterinary Medicine, National Chung Hsing University, 250-1, Kuo Kuang Rd., Taichung City, 402 Taiwan, R.O.C. Corresponding author E-mail: [email protected]
Thai J Vet Med. 2011. 41(1): 111-117.
Shia W. et al. / Thai J Vet Med. 2011. 41(1): 111-117.
บทคัดย่อ ค่าพลาสมา Matrix Metalloproteinase-9 ที่ใช้เป็นค่าบ่งชี้ในเนื้อเยื่อเนื้องอกเต้านมสุนัข Wei-Yau Shia1 Shih-Ming Liu1 Chun-Sheng Lee2 Jian-Liang Lin2 Chih-Chung Chou1 Yumi Yuasa1 ShihChieh Chang1,2 Wei-Ming Lee1, 2* วัตถุประสงค์ของการศึกษา เพื่อเข้าใจการเปลี่ยนแปลงค่า MMP-2 และ MMP-9 ในสุนัขที่ตรวจพบเนื้องอกเต้านม โดยทําการ ตรวจหาค่า MMP-2 และ MMP-9 ในพลาสมา และเนื้อเยื่อเนื้องอกเต้านม และเนื้อเยื่อปกติข้างเคียง โดยวิธี gelatin zymography พบว่า ระดับของค่า MMP-2 และ MMP-9 มีค่าสูงขึ้นอย่างมีนัยสําคัญทางสถิติ เมื่อเปรียบเทียบกับเนื้อเยื่อเต้านมปกติ (p<0.05) ค่าพลาสมา MMP-9 ในกลุ่มมะเร็งเต้านม มีค่าสูงกว่ากลุ่มเนื้องอกเต้านม อย่างมีนัยสําคัญทางสถิติ (p=0.15) และกลุ่มควบคุมปกติ (p<0.05) เช่นเดียวกับค่าพลาสมา MMP-2 ในกลุ่มมะเร็งเต้านม มีค่าสูงกว่ากลุ่มเนื้องอกเต้านมอย่างมีนัยสําคัญทางสถิติ (p=0.54) และกลุ่มควบคุม ปกติ (p<0.01) ค่าพลาสมา MMP-9 มีความสัมพันธ์กับค่า MMP-9 ที่ตรวจพบในก้อนเนื้องอก (r=0.78, p=0.02) แต่ค่า MMP-2 ไม่พบ ความสัมพันธ์ดังกล่าว สรุปผลการศึกษาแสดงถึงค่า MMP-2 และ MMP-9 มีความเกี่ยวข้องกับความรุนแรงของมะเร็งเต้านมในสุนัข และค่า พลาสมา MMP-9 สามารถนํามาใช้ในการพยากรณ์และเฝ้าติดตามโรคเนื้องอกเต้านมในสุนัขได้ คําสําคัญ: สุนัข เนื้องอกเต้านม matrix metalloproteinase 1 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung City, 402 Taiwan, R.O.C. 2 Veterinary Medical Teaching Hospital, College of Veterinary Medicine, National Chung Hsing University, 250-1, Kuo Kuang Rd., Taichung City, 402 Taiwan, R.O.C. *ผู้รับผิดชอบบทความ E-mail: [email protected] Introduction Mammary gland tumor (MGT) is one of the most common neoplasms in female dogs, approximately 40-50% of which are considered malignant (Lee et al., 2004). Tumor recurrence and metastasis are the most important causes of patient death after surgery. The matrix metalloproteinase (MMP) family is thought to be responsible for the accelerated breakdown of extra-cellular matrix (ECM) associated with tumor invasion and metastasis (Kawai et al., 2006). Matrix metalloproteinases (MMP) are a family of zinc-containing, proteolytic enzymes implicated in the degradation of extracellular matrix (Blavier et al., 2006). These proteolytic enzymes have been implicated in a variety of physiologic and pathological conditions. Under physiologic condition, for example, they are expressed by a variety of cell and tissue types and are involved in processes such as trophoblast invasion (Cross et al., 1994), development (Matrisian and Hogan, 1990), tissue remodeling (Salamonsen et al., 1999; Blavier et al., 2006), ovulation (Russell et al., 1995), angiogenesis (Birkedal-Hansen, 1995), wound healing (Madlener et al., 1998), etc. Meanwhile, these proteolytic enzymes are implicated
in the pathologic processes in animals including tumor invasion and metastasis in experimental animals (Sawaya et al., 1998) and humans (Moses et al., 1998; Kleiner and Stetler-Stevenson, 1999). It has been documented that increased MMPs activity are associated with invasion, metastasis and prognosis in human and animal malignancies (Moses et al., 1998; Lana et al., 2000; Hirayama et al., 2002; Loukopoulos et al., 2003; Kawai et al., 2006) such as breast cancer (Talvensaari-Mattila et al., 1998; Dalberg et al., 2000). Recently tissue MMP-2 and -9 were documented in canine tumors and a high level of pro-MMP-9, proMMP-2 and active MMP-2 were detected in most canine tumors. In addition, high levels of MMP-9 activity were found in the sera of canines with mammary adenocarcinoma indicating that MMP-9 plays an important role in the progression of a canine mammary tumor and that serum MMP-9 analysis provides as early diagnosis of adenocarcinoma (Yokota et al., 2001). In humans, plasma MMP-9 is a useful marker in the follow-up and in the assessment of prognosis in breast cancer patients (Farias et al., 2000; Ranuncolo et al., 2003). In this study the zymographic band patterns of MMPs from plasma and mammary gland tissues including tumor and nearby normal tissue were measured by Image J software (National Institutes of Health, USA).
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Therefore, the aim of this study was to find a potential marker from MMPs to reflect the tissue level of mammary glands tumor in dogs.
The tissue and plasma samples were subjected to electrophoresis on 8% SDS-PAGE co-polymerized with gelatin (0.2%) as the substrate. 15 μl of homogenized supernatant were loaded in each well. Besides, 2 μl human MMP-2 marker (Chemicon®, CA) and 10 μl human MMP-9 markerZ (R&D®, USA) were loaded in the same gel. After the electrophoresis was complete, the gel was washed by 200 ml 2.5% Triton X-100 for 1 hour, and then washed by DW several times. The gel was incubated in 10 times diluted digestion buffer (15.6 g Tris-HCl and 1.47 g CaCl2 were dissolved in DW, pH=7.8) at 37oC for 19 hours. The gel was then stained with 2% Coomassie Blue R250 in 50% methanol and 10% acetic acid for 30 min, and destained in 40% methanol and 10% acetic acid, until the active bands became clear.
Materials and Methods Animals: Surgically resected tissues from twelve cases of canine mammary gland tumor were obtained at the Veterinary Medical Teaching Hospital, National Chung Hsing University. Six healthy female beagles were used in this study as a control group for the determination of plasma MMP-2 and MMP-9 activity. The animals were used with the owner’s consent. Histopathologic examination: For histological examination, part of the tumor and nearby normal tissues were fixed in 10% formalin and embedded in paraffin. Thin sections were then prepared and stained with hematoxylin & eosin. The biopsy sections were histopathological diagnosed according to the guideline of World Health Organization (Misdrop et al., 1999) as nine malignant tumors including five malignant mixed type mammary tumors, one fibrosarcoma, three adenocarcinomas and three benign mammary tumors. Tissue specimens: Twelve tumor tissues were clinically classified by TNM system, including two TNM stage II, eight TNM stage Ш, and two TNM stage IV (Rutteman and Kirpensteijn, 2003). Among the 12 MGT cases, five cases (three TNM stage Ш and two TNM stage IV) had bilateral enlarged lymph nodes (retropharyngeal and/or inguinal lymph node) and four cases (TNM stage Ш) had ipsilateral enlarged lymph nodes. Tumor tissues were collected from the inter-zone between the central and margin of the tumor and cut into several 0.3x0.3x0.3 cm size pieces and store in cryogenic freezing tube (Nalgene®, Thermo Fisher Scientific, USA) at -70oC refrigerator. Two hundred mg of each tumor mass were collected and ground in a tissue grinder with 10 ml phosphate buffer saline (PBS, buffer solution. The ground tumor tissues were homogenized at 1,000 revolution for 8 min in a homogenizer（Macro ES Digital Programmable Homogenizer, OMNI, USA）and then ground by hand (Kimbo Kontes, USA) for 5 min. The homogenate was placed into micro-centrifuge tube and centrifuged at 10,500xg for 30 min. The supernatant was collected and kept at -20oC until MMPs determination. All plasma samples were collected by jugular vein puncture from the 12 cases of canine mammary gland tumor and 6 normal spayed beagle bitches for MMPs determination. Additionally, blood samples from adenocarcinoma and one case of malignant mixed mammary tumor before and after mastectomy were collected to observe the change of plasma MMP2 and MMP-9. Gelatin zymography: Gelatin zymography of MMP-2 and MMP-9 from the plasma and tissue was performed as previously described (Gerlach et al., 2005; Souza-Tarla et al., 2005; Gerlach et al., 2007). Briefly, Dual Mini Slab Kit (Model: AE-6450, ATTO, Japan) was used in this study for the electrophoresis.
Measurement of zymographic band patterns: The digestive areas of the gels were photographed. The digested area was measured by Image J (Image Processing and Analysis in Java) software (National Institute of Health, USA). The value depended on the size and brightness of the digestive area. Statistics: Differences in parameters between tumor and normal tissue were evaluated using the MannWhitney U test. Correlation coefficient (r) was calculated between different factors using the Spearman correlation. Values are expressed as mean±SEM. P<0.05 was considered statistically significant.
Results and Discussion The zymographic band pattern of MMP-2 and MMP-9 were presented on the gel. The latent form and active form of MMP-2 were 92 kDa and 86 kDa and of MMP-9 were 68 kDa and 62 kDa, respectively (Fig. 1). There was an increase in both the latent and active form of MMP-9, but only an increase in the potential activity of MMP-2 was found in our study. The difference of the latent form of MMP-2 in
Figure 1 The activity of MMP-2 and MMP-9 in different tissues (A) and plasma (B) in dogs with MGT. Latent form of MMP-2 and MMP-9 were 68 kDa and 92 kDa, respectively. Active form of MMP-2 and MMP-9 were 62 kDa and 86 kDa, respectively. (A) M1: human MMP-9 marker, M2: human MMP2 marker, T: tumor tissue, N: normal tissue, L: lymph node.
Figure 2 The activity of MMP-2 in tumor tissue and nearby normal mammary gland tissue. (A; tumor tissue MMP-2 was significantly higher than that in normal tissue, B: MMP-2 in the malignant tumor tissue was significantly higher than that in the benign tumor tissue.) * indicate significant difference compared to the control (p<0.05); ## indicated significant difference compared to benign tumor tissue (p<0.01)
the tissue and plasma between the tumor and normal sample was not as much as MMP-9. This could be because MMP-9 synthesized in cell cytosol, the enzyme can be stored in either a latent or an active form, which is contrast to MMP-2, which can be stored only in a latent form (Nguyen et al., 2001). The activity of MMP-2 in the tumor tissue (24.86±5.22) was significantly higher (p<0.05) than that in normal tissue (11.22±2.56) (Fig. 2A). In addition, the activity of MMP-2 in the malignant tumor tissue (32.45±6.67) was significantly higher (p<0.05) than that in the benign tumor tissue (11.14±2.09) (Fig. 2B). The proteolytic activity of the malignant tumor tissues balance shifted to a more enzymatic activity, very likely by increased activation or decreased inhibitor level. The result suggested that malignant tissues had higher proteolytic activities which was in agree with the result of Lee et al. (1996). Similar to MMP-2, the activity of MMP-9 in the tumor tissue (86.17±17.27) was significantly higher (p<0.05) than that in the normal tissue (30.14±5.81) (Fig. 3A). The activity of MMP-9 in the malignant tumor tissue (83.34±16.36) was higher than the benign tumor tissue (63.94±13.14) but without significant difference (Fig. 3B). This might be because of the smaller sample sizes and higher standard deviation between individuals. Referring to the result of the level of MMP-2 and MMP-9 in
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Figure 3 The activity of MMP-9 in tumor tissue and nearby normal mammary gland tissue. (A: MMP-9 in the tumor tissue was significantly higher than that in the normal tissue, B; there was no significant difference of the activity of MMP-9 between the malignant tumor tissue and the benign tumor tissue) * indicate significant difference compared to the control (p<0.05); ## indicated significant difference compared to benign tumor tissue (p<0.05)
Figure 4 The activity of plasma MMP-2 in dogs with and without MGT. A: plasma MMP-2 was significantly higher than that in control dogs, B: plasma MMP-2 in cases with malignant tumor tissue and with benign MGT were significantly higher than that in control dogs. ** indicated significant difference compared to the control (p<0.01)
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Figure 6 Correlation of MMP-9 between tumor tissues and plasma samples (B, r=0.78, p=0.02), but not in MMP-2 (A, r=0.15, p=0.68). Figure 5 The activity of plasma MMP-9 in dogs with and without MGT. A: plasma MMP-9 in tumor tissue was significantly higher than that in control dogs, B: there was no significant difference in the activity of plasma MMP-9 between cases with malignant tumor and cases with benign tumor. * indicate significant difference compared to the control (p<0.05); ** indicated significant difference compared to the control (p<0.01)
malignancy of the tumor, the observation was similar to previous studies (Lana et al., 2000; Yokota et al., 2001; Loukopoulos et al., 2003). The mean activity of plasma MMP-2 and MMP-9 in the dogs with MGT (55.58±8.14, 63.55±8.30) were significantly higher (p<0.05) than those in the normal dogs (12.09±1.48, 26.30±3.54), respectively (Fig. 4A and 5A). Likewise, plasma MMP-2 in the dogs with malignant (64.55±14.33) or benign (46.62±7.53) MGT was significantly higher (p<0.05) than that in the control dogs (12.09±1.48). There were no significant differences in activity of plasma MMP-9 between the dogs with malignant MGT (79.34±14.81) and the dogs with benign MGT (47.77±3.37), although the level of plasma MMP-9 in the dogs with benign MGT increased in comparison to the normal dogs. A positive correlation was noticed in MMP-9 between the tumor tissues and the plasma samples (r=0.78, p=0.02), but not in MMP-2 (r=0.15, p=0.68) (Fig. 6). No significant correlation between the tissue and the plasma in MMP-2 was likely to occur because MMP-2 in the benign tumor tissue was did not increase parallel to plasma level. The result might be because MMP-2 antigen levels were not significantly altered in mammary tumor tissue, but active MMP-2 at higher levels in malignant tissue as compared with benign tissue (Hanemaaijer et al., 2000). The result is in accordance with our finding that the malignant tumor
tissue MMP-2 was significantly higher than the benign one. The metastasis of tumor is assumed that the primary mechanism by which MMPs promote tumor metastasis is by the degradation of the ECM. Collagen IV, the main component of basement membrane, is thought to be degraded mostly by MMP-2 and MMP-9. These MMPs may therefore play a critical role in the conversion of in situ breast cancers to invasive lesions (Duffy et al., 2000). Additionally, increased MMP activity in tumor local environment results in proteolytic cleavage of membrane-associated extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) releasing soluble EMMPRIN. Soluble EMMPRIN in turn acts in a paracrine fashion on stroma cells that are both adjacent and distant to tumor sites to further stimulate the production of MMPs and additional EMMPRIN, which consequently contributes to tumor angiogenesis, tumor growth, and metastasis (Tang et al., 2004). Besides the regulation of EMMPRIN on MMP-2 and MMP-9, MMP-9 is secreted by stromal cell and inflammatory cells including neutrophils, macrophage and mast cell (Deryugina and Quigley, 2006). In our study, lymphocyte was the prominent inflammatory cell, especially in malignant MGT. Neutrophil was also found in tumor tissue but not correlated with the malignancy of MGT. In addition, inflammatory cytokines were proved to recruit different inflammatory cells, for example, neutrophils recruited by IL-2, eosinophils recruited by IL-4 and IL-5, natural killer cells recruited by IL-12, and macrophages recruited by IFN-γ (Cavallo et al., 1992; Cavallo et al., 1997; Musiani et al., 1997; Di Carlo et al., 1998). The high levels of MMP-2 and MMP-9 presented in this study indicated the level of tumor
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malignancies which may be associated with the angiogenesis and inhibition of immune cell proliferation. In the micro-environment of a tumor, high levels of MMP-2 and MMP-9 are associated with the angiogenesis (Deryugina and Quigley, 2006), an important process in tumor metastasis, growth and tumor cells entering general circulation (Woodhouse et al., 1997). In addition, the expression of vascular endothelial growth factor (VEGF) can be affected by the MMP-9 during angiogenesis (Folgueras et al., 2004). Besides the angiogenesis, MMP-2 and MMP-9 can inhibit the proliferation of T-cells allowing tumor cells to escape immune surveillance (Sheu et al., 2001). Based on our knowledge there are few publications concerning the relationship between plasma MMP-2 and MMP-9 level and malignancy of tumor. In humans, it has been documented that high plasma MMP-9 levels are present in patients with lung cancer, but no association has been reported with the tumor stage and malignancy (Farias et al., 2000). Nevertheless, the plasma level of MMP-9 has a positive correlation with the status of patients with breast cancer (Ranuncolo et al., 2003). Thus, the implementation of plasma MMP-2 and MMP-9 levels as a useful parameter in correlation with tumor malignancy is warranted. Though high plasma level of MMP-2 and MMP-9 were observed in dogs with malignant MGT in this study, only plasma MMP-9 levels reflected the activity of MMP-9 from MGT tissues. Taken together, plasma MMP-9 analysis is a potential early diagnosis method in the determination of canine MGT progression. In conclusion, the activity of tissue MMP-2 and MMP-9 was positively correlated with the malignancy of MGT in dogs. The activity of tumor tissue MMP-9 was positively correlated with the activity of plasma MMP-9 in dogs with MGT, but not MMP-2. Although the sample size was small in this preliminary study, plasma MMP-9 level might be suggested as a useful marker in the follow-up and in the assessment of prognosis in dogs with MGT. However, it still needs further investigation.
Acknowledgements The authors would like to thank the kind help on the critical reading and writing by Dr. Amelia M. M. Jack.
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Tracing IV - 50 mm/sec History A nine years old male Golden retriever weighing 32.5 kg came to the animal hospital, Chulalongkorn University with a history of walking difficulty and lameness for the last 3 months. After the weakness of both hind limbs started, it then progressed to the fore limbs. He also had difficulty in controlling urination. By physical examination, the dog had normal heart sound with arrhythmia. The tetraparesis was detected with muscular atrophy especially at the hamstring. The complete blood count, liver enzyme (ALT) and plasma creatinine concentration were normal. Wobbler syndrome with osteoarthritis was diagnosed. Thoracic radiograph
showed normal heart size and shape and mild diffused interstitial and bronchial pattern of lung. The electrocardiography was performed as seen in the strip I and II. Echocardiography showed mild mitral valve regurgitation and mild systolic dysfunction. Blood pressure was measured with a systolic of 128 and diastolic of 86 mmHg using oscillometric technique. Dog was on doxycycline and prednisolone and came back 2 weeks later with clinical improvement and walked by himself. The ECG was repeated 2 and 4 weeks later and shown in tracing III and IV, respectively. Please answer before turning to the next page.
of Physiology, 2Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University *Corresponding author
Buranakarl C. and Chansaisakorn W./ Thai J Vet Med. 2011 41(1): 119-120.
Interpretation Tracing I, II– Accelerated ventricular rhythm with premature ventricular complex Tracing III – Respiratory sinus arrhythmia Tracing IV – Respiratory sinus arrhythmia
Tracing II Accelerated ventricular rhythm is commonly seen in dogs with or without primary heart disease. It may be seen in dogs with trauma, gastric torsion, splenic tumor, etc. The heart rate in tracing I was approximately 140 beats/minute. The complexes with ventricular in origin are not a paroxysmal tachycardia although the transition from sinus to ventricular and back occurs abruptly but this term denotes an abrupt change in heart rate. There was a slowing of the sinus rhythm which may be due to respiratory sinus arrhythmia below the discharge rate of the VPCs, thereby removing the overdrive suppression by the faster sinus beats and allowing the ectopic focus to reach the threshold potential and initiate a ventricular response (solid dots). When the sinus rhythm accelerates and exceeds the rate of ventricular rhythm,
it again overdrives the ventricle and prevents the ectopic focus from reaching its threshold potential. The fusion beats (opened arrows) were seen in tracing II with a typical in which the P-wave have a shorter PR interval and the morphology of fusion beat is intermediate between the sinus and ectopic beats. In tracing I, one different VPC was noticed (arrow) which had bizarre shape and emerged very early. Therefore, multiple sites of ventricular ectopic foci were found. The ECG recorded at 2 and 4 weeks later showed normal sinus arrhythmia. These findings confirm that the electrical instability of the heart was not related to the organic disease of the heart but may be rather due to other factor including the disease associated with pain.
Ophthalmology Snapshot Nalinee Tuntivanich
History A 7 year-old male Pug had had bilateral mucopurulent ocular discharge for several months. He was referred from a private clinic to the Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University for ocular treatment. From ophthalmic examination, STT 1 value was 10 and 5 mm on the right and left eye, respectively. Bilateral pigmentary keratitis was present with more prominent sign on the left eye compared to the other. On the right eye, a 5 mm, light pink, oval mass was noticed on the upper bulbar conjunctiva close to corneal limbus. No bleeding was observed. After the dog had prescriptively been on topical corticosteroid and topical cyclosporine for 9 months, STT level increased while size of the mass was reduced to 3 mm in diameter. The mass was surgically excised and submitted for histopathological investigation. No evidence of recurrence had been noted after 2 years of surgery.
Figure 1 Photographs of the right eye of the Pug in the (a) front view and (b) oblique view demonstrating conjunctival mass. (For better quality, figures can be viewed in the TJVM website)
Question Give the differential diagnosis of the conjunctival mass based on clinical appearance.
Please turn to the next page for answers ………. Ophthalmology Clinic, Department of Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
Tuntivanich N./ Thai J Vet Med. 2011 41(1): 121-122.
Figure 2 Front-viewed images of (a) an actual photograph and (b) a schematic drawing of the right eye, illustrating structures of the eye and conjunctival lesion. Answers Ophthalmic differential diagnosis: 1. Plasmoma 2. Squamous cell carcinoma 3. Hemangioma Final diagnosis: Conjunctival hyperplasia with lymphoplasmacytic conjunctivitis Comments Due to its clinical presentation, this lesion is called a non-neoplastic conjunctival mass. Lymphoplasmacytic/plasmacytic conjunctivitis (or plasmoma) is common in brachycephalic breeds (including Pug) that were predisposed to chronic superficial keratitis (CSK). This abnormality is associated with immune-mediated inflammatory condition across the ocular surface; subconjunctiva, cornea, limbus, and third eyelid. Conjunctiva is usually thicken and hyperemic with a majority of lymphocytes and plasma cells, histopathologically. In addition to a limitation of CSK factors, immunosuppressive drug with or without topical corticosteroid can be used to control progression and possibly reduce the lesion. Even though the lesion may recur following excision, excision with or
without cryotherapy is still considered a successful treatment. Conjunctival squamous cell carcinoma is uncommon in dogs. If present, it usually occurs as an elevated, soft, pink mass on the bulbar conjunctiva at limbus. Affected dogs, which are brachycephalic breeds, mostly have history of chronic keratitis. Exposure to UV light is thought to be important factor. Biopsy can be performed for final diagnosis. Hemangioma is clinically presented as a bright red, friable, bleeding mass on temporal bulbar conjunctiva. Dogs kept out door with greater exposure to UV light tend to be predisposed to this neoplasia. Biopsy can be performed for final diagnosis. References Dubielzig, R.R, Ketring, K.L., McLellan, G.J. and Albert, D.M. 2010. Diseases of the eyelids and conjunctiva. In: Veterinary Ocular Pathology; a comparative review. Philadelphia: Saunders Elsevier. 166-190. Read, R.A. 1995. Treatment of canine nictitans plasmacytic conjunctivitis with 0.2 percent cyclosporine ointment. J Small Anim Pract. 36(2): 50-56.
ULTRASOUND DIAGNOSIS Phiwipha Kamonrat
A thirteen-year-old, spayed female, Shih-tzu dog was presented at the Chulalongkorn University, Small Animal, Veterinary Teaching Hospital for an evaluation of a progressive abdominal enlargement with an increasing liver enzyme for more than a year. The dog had recently showed clinical signs of polyuria, polydipsia and polyphagia. Physical examination revealed pink mucous membranes and a pendulous abdomen without pain on palpation. A routine blood work, urinalysis and radiographic examination were performed. Abnormal clinical values included elevation of serum ALT (225 IU) and ALP (1,431 IU). Survey abdominal radiographs demonstrated hepatomegaly. Ultrasonography of the liver and adrenal glands was performed to obtain more specific information.
Sagittal and transverse scans of the liver showed enlargement with rounding of the caudoventral edge. The overall liver was isoechoic to the spleen and hyperechoic to the kidneys and falciform fat (Fig. 1A and 2A). The echogenic portal vein margin appeared less prominent compared with the hepatic parenchyma. There were multifocal, hypoechoic, circumscribed, 3-25 mm in diameter scattered throughout the liver. The right and left adrenal glands measured 5 and 7 mm in thickness, respectively. The upper normal limit of the left adrenal size together with the clinical findings and laboratory work-up in this dog were consistent with a hyperadrenocorticism. Ultrasonography of other abdominal organs appeared normal in echotexture. A fine needle aspirate was taken and a cellular diagnosis of hydropic and fatty degeneration-related steroid hepatopathy was suggested. Diagnosis Ultrasonographic hepatopathy.
Department of Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
Kamonrat P./ Thai J Vet Med. 2011 41(1): 123-124.
Figure 1 Ultrasonographic images of a thirteen-year-old, spayed female, Shih-tzu dog, in dorsal recumbency. The increased echogenicity of overall liver was hyperechoic to the falciform fat. The echogenic portal vein wall was less prominent due to the relative increase in hepatic parenchymal echogenicity. Multifocal hypoechoic nodules of variable size were diffuse throughout the liver.
Figure 2 Schematics of the relative positions of the structures scanned in figure 1. L: liver, F: falciform fat, P: portal vein, N: nodule, S: stomach.
Comments Ultrasonography is a valuable method for evaluating the hepatic parenchymal abnormalities in animals. Diffuse disorders are more difficult to detect than focal processes because they cause less distortion of normal hepatic architectural landmarks. The ultrasonographic evaluation of liver size is usually based on subjective assessment. Hepatomegaly is generally easier to image, has a large volume between the diaphragm and stomach, extends ventrally to the stomach and has rounding of the ventrocaudal margin. Hepatic lipidosis, steroid hepatopathy, lymphosarcoma and hepatitis are more common in a hepatomegaly (d’ Anjou, 2008). Diffuse parenchymal hepatic disease appears ultrasonographically as a change in overall hepatic echogenicity. It can be increased, reduced or unaffected. At the same scanning depth and instrument gain settings, echogenicity of liver is considered increased if the echogenicity of the hepatic parenchyma is greater or similar to the spleen and greater than the renal cortex and falciform fat. The margins of portal veins may be less prominent owing to increased hepatic echogenicity. Diseases that
commonly cause an increase in hepatic echogenicity include fatty change, steroid hepatopathy and cirrhosis (Biller et al., 1992). Fatty infiltration of the liver may cause a fine, diffusely increased echogenicity and hepatomegaly. Steroid hepatopathy can cause both hepatomegaly and diffusely increased echogenicity in the liver. These changes have been noted with both hyperadrenocorticism and iatrogenic Cushing’s disease. Limitations of ultrasonography include lack of specificity for focal or multifocal hepatic lesions and insensitivity for diffuse disease. A more definite diagnosis is usually based on information obtained from the history, clinical signs, physical examination, laboratory data, ultrasonography and cytological or histopathological results. References Biller D.S., Kantrowitz B. and Miyabayashi T. 1992. Ultrasonography of diffuse liver disease: A review. J Vet Int Med. 6(2): 71-76. d’ Anjou, M.A. 2008. Liver In: Atlas of Small Animal Ultrasonography. D. Pennink and M.A. d’ Anjou (eds.) Ames: Blackwell Publishing. 244-261.
WHAT IS YOUR DIAGNOSIS Pranee Tuntivanich Suwicha Chuthatep Signalment An 11-year-old female spayed mixed breed dog History There was a small mass (approximately 4 cm length) above the cranial lumbar area. This dog had presented with both hind limb paralysis during the past 3 weeks without traumatic history. She could not control both urination and defecation. It was painful when touching on her back.
Clinical Examination She lost all her spinal reflexes in both hind limbs. Thrombocytopenia and increasing of the serum alkaline phosphates were detected from blood examination. Negative result from bacterial culture examination was shown in sample from lumbar mass aspiration. Radiographic Examination Plain ventrodorsal and right lateral radiographs of the lumbar vertebral column were taken to evaluate the vertebral alignment and other spinal bone abnormalities.
Give your diagnosis and turn to the next page. Department of Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
Tuntivanich P. and Chuthatep S. / Thai J Vet Med. 2011. 41(1): 125-126.
Ventrodorsal and right lateral vertebral radiographs (Fig. 1, 2) revealed an increase of radiolucency of the 6th lumbar vertebrae. The lumbar vertebras were in normal alignment. On lateral radiograph (Fig. 2), an obvious pathological fracture of the 6th lumbar vertebrae was detected as a decrease of the vertebral body length, partial loss of vertebral laminar and severe bone destruction. Similarity in the 2nd lumbar vertebrae also presented severe bone destruction of the spinous process and caudal articular facet (Fig. 3). Moreover, the osteophyte formation can be seen between the ventral portion of the 7th lumbar vertebrae and sacral spine.
Spinal tumor can occur in various locations of the spine including spinal body, spinal canal, dural sac and spinal cord. The tumor can primarily be originated from the spine or be metastatic in the same frequency. Spinal tumor usually causes spinal bone destruction, ruptured intervertebral disks, spinal canal and spinal cord compression which typically produce progressive pain and disability. The thoracic and lumbar spines are the most frequent sites for spinal tumors. Primary and secondary spinal tumors often extend into adjacent vertebrae which are different from long bone tumors that do not normally cross joint spaces. Additionally, there is no relation between the degree of spinal bone destruction from radiograph and the degree of neurologic deficit in animal. Myelography may be used to indicate a location of the definite lesion of the spine but the cell type cannot be identified. In cases of unclear spinal tumors, computed tomographic examination can be performed to investigate small areas of bone destruction or bone deposition.
Radiographic diagnosis Spinal tumor
Figure 3 Severe bone destruction in spinal column (white arrows)
References Farrow, C.S. 2003. Spinal tumors (vertebral tumors) and tumor-like lesions. In: Veterinary Diagnosis Imaging the Dog and Cat. St. Louis, Missouri: Mosby. 310-318.